Chromosome conformation analysis

ABSTRACT

Disclosed herein are compositions, methods and kits for analyzing three-dimensional chromatin and/or chromosome conformation. Method are also disclosed for using the methods disclosed herein for diagnosing diseases such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 13/475,089, filed May 18, 2012, which claimspriority to U.S. Provisional Application No. 61/487,614, filed May 18,2011, U.S. Provisional Application No. 61/579,444, filed Dec. 22, 2011and U.S. Provisional Application No. 61/579,876, filed Dec. 23, 2011which disclosures are herein incorporated by reference in theirentirety.

FIELD

This disclosure relates to methods, reagents and kits useful forepigenetic analysis of DNA.

BACKGROUND

Epigenetics is the study of heritable changes in gene function that donot involve changes in DNA sequence. These changes can occur due to thechemical modification of specific genes or gene-associated proteins ofan organism. These modifications can affect how genes are expressed andused in cells. For example, methylation of specific regions in genesequences, such as CpG sites, can make these genes lesstranscriptionally active. Another example is post-translationalmodification of histone proteins around which genomic DNA is wound. Thishistone modification can affect the unwinding of the DNA duringtranscription which, in turn, can affect the expression of thetranscribed genes. Furthermore, chromosome conformation or chromatincompaction can also have an effect on gene expression, such as affectingthe accessibility of the template to polymerases or changing theproximity between genes and genomic sequences.

Important chromosomal activities and gene expression have been linkedwith the structural properties of the chromosome, such as their spatialconformation. Furthermore, the local properties of chromatin fibers havealso been shown to influence gene expression. Higher order structures ofchromatin, such as 30 nm fibers, chromatin loops and axes, andinterchromosomal connections have also been shown to play a role in geneexpression and recombination.

Epigenetic mechanisms are essential for normal development andmaintenance of the normal gene expression pattern in many organismsincluding humans. Recent studies suggest that epigenetic alterations maybe the key initiating events in some forms of cancers and global changesin the epigenome are a hallmark of cancers. Epigenetic mechanisms thatmodify chromatin structure can be divided into four main categories: 1)DNA methylation, 2) covalent histone modifications and noncovalentmechanisms such as incorporation of histone variants, 3) nucleosomeremodeling and 4) non-coding RNAs which include microRNAs (miRNAs). Therole of DNA methylation and histone modifications in cancer initiationand progression is well established; however, the changes in chromatinstructure that accompany DNA methylation and histone modifications areless well understood

The analysis of chromosomal conformation has been complicated bytechnical limitations. For example, analysis by electron microscopy islaborious and cannot be used to view specific loci;fluorescently-labeled DNA binding proteins permit the visualization ofspecific loci, but only a few loci can be examined simultaneously.Fluorescence in situ hybridization (FISH) analysis can examine multipleloci, but the severe experimental conditions may adversely affectchromosomal organization.

In an attempt to overcome the limitations of visual chromosomalanalysis, methods using the polymerase chain reaction (PCR) have beendeveloped. For example, the chromosome conformation capture (3C) methodanalyzes overall chromosomal spatial organization and physicalproperties at a higher resolution (see, Dekker et al., Science295:1306-1311 (2002)). In 3C experiments, genomic DNA (gDNA) andproteins in the chromosomes are fixed in place by cross-linking. Thecross-linked gDNA is digested by a restriction enzyme and ligated beforebeing purified for analysis. Physical interactions between genomic lociare identified as specific cross-ligated DNA elements using PCRamplification. As a result of 3C methods, spatial information isconverted to quantifiable DNA sequences. However, the wide adaptation of3C methods has been hindered by the lack of quantitative processcontrols and cumbersome protocols.

Derivative methods of 3C, such as 4C and 5C, have also been developed.4C and 5C differ from 3C only in the analysis of the ligation product.In 4C, the ligation product is first amplified by PCR using twooutwardly facing primers from the restriction site to create a circularDNA molecule which is then analyzed by microarray technology. In 5C, theligation products are mixed with special primers designed to anneal atthe ends of the restriction fragment. Analysis is carried out either byuse of a microarray or by sequencing against a 3C library of ligationproducts.

However, there are several disadvantages associated with these 3C-basedmethods. The assays are time consuming and are not precise and sensitiveenough for reactions with low digestion or ligation efficiency. Thesemethods also require significant dilution of the DNA sample. As aresult, large quantities of the sample may be needed, which may notalways be available. In addition, the 3C-based methods listed abovesuffer from low throughput and are unable to solve the entire spatialarrangement of the chromatin in the nucleus and therefore must focus oncapturing interaction partners of a limited number of loci. The presentteachings overcome these and other disadvantages and limitations and areuseful for capturing interaction partners in the different regions ofchromatin in an unbiased fashion.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several exemplary embodiments ofthe disclosure and together with the description, serve to explaincertain teachings. The skilled artisan will understand that thedescribed drawings are for illustration purposes only. The drawings arenot intended to limit the scope of the present teachings in any way.

FIG. 1: A schematic representation of the 3C method according toembodiments of the present teachings.

FIG. 2: A schematic representation of the restriction and ligationcontrol assays according to embodiments of the present teachings.

FIG. 3: A schematic representation of a method of analyzing non-specificligation according to an embodiment of the present teachings.

FIG. 4: A schematic representation of chromosomal conformation analysisand universal library preparation using linear half-adaptors with a Toverhang, according to an embodiment of the present teachings.

FIG. 5: A schematic representation of chromosomal conformation analysisand universal library preparation using 5′-phosphate looped adaptorswith cohesive ends, according to an embodiment of the present teachings.

FIG. 6: A schematic representation of chromosomal conformation analysisand universal library preparation using 5′-OH looped adaptors with a Toverhang, according to an embodiment of the present teachings.

FIGS. 7A and 7B: A schematic representation of universal 3C libraryconstruction to be used in the methods disclosed herein, according to anembodiment of the present teachings.

FIG. 8: Shows the locations of the digestion and ligation control assayprobes and primers in a gene desert region as described in the presentteachings. ENr313 is located in chromosome 16 (SEQ ID NO: 1).

FIG. 9: Graphical depiction of quantitative measurement of restrictionefficiency using the methods described herein (see Example 2).

FIG. 10: Effects of reducing ligation volume on between-cell ligationevents using the methods disclosed herein (see Example 3).

FIG. 11: A schematic of assay design and interaction detection forβ-globin locus control region (LCR) (see Example 4).

FIG. 12: A schematic summary of LCR interaction mapping (see Example 4).

FIG. 13: A graphical representation of interaction frequency vs.distance as determined using the methods of the embodiments disclosedherein (see Example 4).

FIG. 14: A schematic diagram of three-dimensional interactions betweenLCR and β-globin genes as determined using methods of the embodimentsdisclosed herein (see Example 4).

FIG. 15: Capture of the LCRE27-14 Interaction and comparison of thesensitivity of the interactions in the library created using the methodsof the present teachings (NGS Library) compared to that of a 3C Library(see Example 5).

FIG. 16: Formation of a chromosome conformation library using themethods according to the embodiments disclosed herein for analysis usingnext generation sequencing methods (see Example 6).

FIG. 17: A diagrammatic representation of phospholipase D 1 (PLD1) genesand assay designs according to embodiments of the present teachings.

FIGS. 18A and 18B: Graphical representation of PLD1 Intron 1 andPromoter 2 long range cis fragments interaction frequency comparison inMCF-7 and MDA-MB-231 cells.

FIG. 19: Graphical representation of PLD1 methylation (FIG. 19A) andexpression (FIG. 19B) in MCF-7 and MDA-MB-231 cells.

FIGS. 20A and 20B: Graphical representation of a comparison of cancermarker genes and microRNA in MCF-7 and MDA-MB-231 cells.

FIG. 21: Graphical representation of MIR21 (FIG. 21B) and targeted tumorsuppressor gene BCL2 (FIG. 21A) expression and CpG methylation in MCF-7and MDA-MB-231 cells.

SUMMARY

Provided herein are methods, compositions and kits that are useful foranalyzing the physical interactions between various genomic elementsthat affect gene regulation, DNA replication and genome organization.Through structures such as loops and bridges, chromatin fibers makemultiple physical contacts with genetic elements that can be tens tohundreds of kilobases apart or more. To study these contacts,specifically to analyze the frequency of interaction and proximitybetween any two genomic loci, chromosome conformation capture methodsprovided herein may be used. The chromosome conformation capture methodsprovided herein convert physical interactions into unique ligationproducts. The concentration of an individual ligation product iscorrelated to the frequency of looping between the two genomic regions.The abundance of the ligation products may be quantified by methods suchas the polymerase chain reaction (PCR), including endpoint PCR,real-time PCR, quantitative PCR (qPCR) and digital PCR (dPCR), and DNAsequencing methods, including fragment analysis, Sanger sequencing, andnext-generation sequencing (NGS), including but not limited to,sequencing by ligation (e.g., SOLiD sequencing), proton ionsemiconductor sequencing, DNA nanoball sequencing, single moleculesequencing, and nanopore sequencing.

The methods disclosed herein overcome the disadvantages of previouslypublished 3C methods and have the following unexpected advantages: 1)quantitative control assays for restriction digestion, ligation andinteraction frequency normalization to allow for precise analysis of theefficiency and quality of the assay; 2) standardized and reproducibleconditions and reagents resulting in more efficient and controlledcross-linking, lysis and restriction digestion; 3) improved ligation; 4)increased purity and yield resulting from a rapid and non-toxicpurification process that improves DNA quality and increases DNArecovery by an order of magnitude; and 5) time savings resulting from astreamlined workflow reducing the time to result by 50%, as compared topublished protocols.

One embodiment provides a method of chromosome conformation analysis(see, FIG. 1), the method including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction endonuclease;

e) ligating the digested DNA by incubating with a ligating agent therebycreating a ligation product;

f) reversing the cross-linking;

g) purifying the ligated DNA; and

h) analyzing the ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the purifying step includes purifying the ligationproduct using column purification. In another embodiment, the purifyingstep includes incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is collected bycentrifugation or by the addition of a magnetic field. The boundligation product is then eluted from the magnetic beads.

Another embodiment provides a chromosome conformation analysis methodthat includes a control assay that monitors the undigested template DNA,herein denoted “Assay 1.” (see, FIG. 2) Assay 1 uses oligonucleotideprimers that specifically hybridize to regions in the target DNA eitherupstream (herein denoted “forward primer”) or downstream (herein denoted“reverse primer”) of the restriction endonuclease cutting site. If therestriction site is not digested, the sequence will be amplified. Anoligonucleotide probe that hybridizes to a sequence upstream of therestriction site may be used to monitor the presence of the undigestedtemplate DNA. In a preferred embodiment, the restriction endonucleaserecognizes a six-base cutting sequence. In a more preferred embodiment,the restriction endonuclease is selected from EcoRI and HindIII.

Yet another embodiment provides a chromosome conformation analysismethod that includes a control assay that monitors the digested templateDNA, herein denoted “Assay 2.” (see, FIG. 2) Assay 2 usesoligonucleotides, herein referred to as “bridge oligos,” that have ablocked 3′ end, a template DNA binding region that hybridizes to the 3′end of the digested DNA, and a 5′ region that hybridizes to primer andprobe sequences. In the first cycle of PCR, the bridge oligo anneals tothe template DNA via the template binding region and the bridge oligosequence is amplified resulting in a first amplification product. In thesecond cycle of PCR, first amplification product is incubated with aforward primer that hybridizes to the 5′ end of the bridge oligo thatcontains the primer binding site which is upstream of the restrictionendonuclease cutting site, and a reverse primer that hybridizes to thedigested DNA which contains the complement of the bridge oligo, andcorresponds to a region downstream of the restriction endonucleasecutting site. If the template DNA is digested, the bridge oligo will beamplified. An oligonucleotide probe that hybridizes to a sequence thatcontains the restriction endonuclease cutting site, which is containedwithin the bridge oligo, may be used to monitor the presence of thedigested DNA. In a preferred embodiment, the restriction endonucleaserecognizes a six-base cutting sequence. In a preferred embodiment, therestriction endonuclease is selected from EcoRI and HindIII.

Another embodiment of the chromosomal conformation methods disclosedherein provides a method for analyzing non-specific between-cellligation (see, FIG. 3), the method including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) dividing the digested DNA into two separate aliquots resulting in afirst aliquot and a second aliquot;

g) ligating a first half-adaptor to the digested DNA of the firstaliquot thereby forming a first ligation product and a secondhalf-adaptor to the digested DNA of the second aliquot thereby forming asecond ligation product, wherein the first and second half-adaptors aredifferent from each other and each half-adaptor contains anon-palindromic overhang on one end and a T overhang on the other end,wherein the non-palindromic overhang of the first half-adaptor iscomplementary to the non-palindromic overhang of the secondhalf-adaptor;

h) combining the first and second ligation products;

i) ligating the first and second ligation products thereby forming athird ligation product; and

j) detecting and/or quantitating the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is collected bycentrifugation or by addition of a magnetic field. The bound ligationproduct is then eluted from the beads.

Another embodiment provides a method for determining thethree-dimensional arrangement of chromatin in a cell (see, FIG. 4), themethod including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) ligating a first half-adaptor to the digested DNA thereby forming afirst ligation product and a second half-adaptor to the digested DNAthereby forming a second ligation product, wherein the first and secondhalf-adaptors are different from each other and each half-adaptorcomprises a non-palindromic overhang on one end and a T overhang on theother end, wherein the non-palindromic overhang of the first adaptor iscomplementary to the non-palindromic overhang of the second adaptor;

g) phosphorylating the first and second ligation products;

h) ligating the first and second ligation products to form a thirdligation product; and

i) analyzing the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors have a stem-loopstructure, herein referred to as “looped half-adaptors.” In yet anotherembodiment, one of the first and second half-adaptors is a linearhalf-adaptor and the other is a looped half-adaptor. In yet a furtherembodiment, one of the first and second half-adaptors is conjugated tobiotin.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is collected bycentrifugation or by addition of a magnetic field. The bound ligationproduct is then eluted from the beads.

Yet another embodiment (see, FIG. 5) provides a chromosome conformationmethod including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) incubating the digested DNA with a phosphatase;

f) ligating a first looped half-adaptor to the digested DNA therebyforming a first ligation product and a second looped half-adaptor to thedigested DNA thereby forming a second ligation product, wherein thefirst and second looped half-adaptors are different from each other andeach looped half-adaptor comprises a stem-loop structure, wherein the 5′end of the stem comprises a cohesive (or non-palindromic) overhang thatterminates in a 5′-phosphate group;

g) heating the first and second ligation products thereby allowing thecohesive ends of each looped half-adaptors to anneal to each other;

h) phosphorylating the free ends of each of the looped half-adaptors;

i) ligating the looped half-adaptors to the DNA fragment thereby forminga third ligation product; and

i) analyzing the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase. In one embodiment, the heating andannealing step is replaced by a nick translation step.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors are loopedhalf-adaptors. In yet another embodiment, one of the first and secondhalf-adaptors is a linear half-adaptor and the other is a loopedhalf-adaptor. In yet a further embodiment, one of the first and secondhalf-adaptors is conjugated to biotin.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is collected bycentrifugation or by the addition of a magnetic field. The boundligation product is then eluted from the beads.

Yet a further embodiment (see, FIG. 6) provides a chromosomalconformation method including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) ligating a first looped half-adaptor to the digested DNA therebyforming a first ligation product and a second looped half-adaptor to thedigested DNA thereby forming a second ligation product, wherein thefirst and second looped half-adaptors are different from each other andeach looped half-adaptor comprises a stem-loop structure, wherein the 5′end of the stem comprises a 5′-OH group and the 3′ end of the stemcomprises a 3′ T overhang;

g) heating the first and second ligation products thereby allowing eachof the looped half-adaptors to anneal to each other;

h) phosphorylating the free ends of each of the looped half-adaptors;

i) ligating each of the half-adaptors to the DNA fragment therebyforming a third ligation product; and

i) analyzing the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase. In one embodiment, the heating andannealing step is replaced by a nick translation step. In oneembodiment, the filling-in step is replaced by a phosphatase step. Inanother embodiment the phosphorylation and ligation steps are replacedby a nick translation step.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors are loopedhalf-adaptors. In yet another embodiment, one of the first and secondhalf-adaptors is a linear half-adaptor and the other is a loopedhalf-adaptor. In yet a further embodiment, one of the first and secondhalf-adaptors is conjugated to biotin.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is collected bycentrifugation or by the addition of a magnetic field. The boundligation product is then eluted from the beads.

Another embodiment provides a method of creating a library forchromosome conformation analysis (see, FIG. 7), the method including thesteps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction endonuclease;

e) filling-in and A-tailing the free ends of the digested DNA;

f) optionally methylating the digested DNA;

g) ligating a first half-adaptor to the digested DNA thereby forming afirst ligation product and a second half-adaptor to the digested DNAthereby forming a second ligation product, wherein the first and secondhalf-adaptors are different from each other and each half-adaptorcomprises a non-palindromic overhang on one end and a T overhang on theother end, wherein the non-palindromic overhang of the first adaptor iscomplementary to the non-palindromic overhang of the second adaptor,further wherein the first or the second half-adaptor is biotinylated;

h) phosphorylating the ligated half-adaptors;

i) nick ligating the first and second ligation products, thereby forminga third ligation product;

j) reversing the cross-linking;

k) purifying the third ligation product;

l) digesting the third ligation product with a restriction endonuclease;

m) filling-in and A-tailing the free ends of the digested DNA;

n) isolating the digested DNA using streptavidin beads;

o) ligating the digested DNA with sequencing primers;

p) analyzing the DNA.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme is selected from a Type IIS orType III restriction endonuclease. In a preferred embodiment, therestriction enzyme is selected from MmeI and EcoP15I. In anotherembodiment, the restriction enzyme yields a blunt end. In anotherembodiment, the filling-in step uses Klenow (exo−) DNA polymerase.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors are loopedhalf-adaptors. In yet another embodiment, one of the first and secondhalf-adaptors is a linear half-adaptor and the other is a loopedhalf-adaptor.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is collected bycentrifugation or by the addition of a magnetic field. The boundligation product is then eluted from the beads.

A further embodiment provides kits for analysis of chromosomeconformation, the kits comprising in one or more separate containers: across-linking agent, a lysing solution, a DNA ligase, and a protease. Inanother embodiment, the kit further provides a cross-linking quencher.In yet another embodiment, the kit further provides magnetic beads, aDNA binding solution, a DNA washing solution, and a DNA elutionsolution. In yet another embodiment, the kit further provides a proteaseinhibitor cocktail, a restriction enzyme stop solution, and aneutralization solution. In another embodiment, the kit comprisescontrol assays, wherein the control assays include Assay 1 and Assay 2,and a first and second half-adaptor. In a further embodiment, the kitsdisclosed herein may further comprise one or more of the following: abuffer, such as phosphate buffer saline, and RNase A. In a furtherembodiment, the kits disclosed herein may further comprise instructionsfor carrying out the methods disclosed herein.

A further embodiment provides for the use of any of the chromosomalconformation analysis methods disclosed herein in the diagnosis ofdiseases, for example, cancer, including but not limited to breastcancer, prostate cancer, lung cancer, skin cancer, cancers of thereproductive tract, brain cancer, liver cancer, pancreatic cancer,stomach cancer, blood cancers (e.g., leukemia and lymphoma), sarcomas,melanomas, and the like; cardiovascular diseases; autoimmune diseasesand disorders; and metabolic diseases and disorders. Another embodimentprovides for the use of any of the chromosomal conformation analysismethods disclosed herein in the diagnosis or determination ofresponsiveness to drugs and medical treatment.

Other embodiments and illustrative aspects, features and advantages ofthe present disclosure will become apparent from the following detaileddescription. It should be understood that the detailed description andthe specific examples, while indicating preferred embodiments, are givenby way of illustration only, since various changes and modificationswithin the spirit and scope of the present disclosure will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION

Provided herein are methods, compositions and kits that are useful foranalyzing the physical interactions between various genomic elementsthat affect gene regulation, DNA replication and genome organization.Through structures such as loops and bridges, chromatin fibers makemultiple physical contacts with genetic elements that can be tens tohundreds of kilobases apart or more. To study these contacts,specifically to analyze the frequency of interaction and proximitybetween any two genomic loci, chromosome conformation capture methodsprovided herein may be used. The chromosome conformation capture methodsprovided herein convert physical interactions between genomic loci intounique ligation products and the concentration of a individual ligationproduct is correlated to the frequency of looping between the twogenomic regions. The abundance of the ligation products can bequantified by methods such as the polymerase chain reaction (PCR),including endpoint PCR, real-time PCR, quantitative PCR (qPCR) anddigital PCR (dPCR), and sequencing methods, including, but not limitedto, fragment analysis, dideoxy sequencing (i.e., Sanger sequencing),Maxam-Gilbert chain termination sequencing, dye terminator sequencing,dye primer sequencing, pyrosequencing, next generation sequencingmethods including, high-throughput sequencing, including massivelyparallel signature sequencing, SOLiD sequencing (e.g., sequencing byligation), sequencing by hybridization, proton ion semiconductorsequencing, DNA nanoball sequencing, single molecule sequencing, andnanopore sequencing.

Eukaryotic genomes are organized non-randomly in the nucleus. Thearrangement of genomic loci not only depends on cell stage and celllineage, but also plays an important role in gene regulation. Thestudies of genome organization and DNA interaction have relied on theChromosome Conformation Capture (3C) method (Dekker et al, supra);however, its applications are hindered by many challenges including: (1)non-optimized and variable results; (2) reagent quality is notcontrolled; (3) large reaction volumes; (4) the lack of processcontrols; (5) the lack of a quantitative readout; and (6) the processitself takes at least five days. The protocols and reagents for keysteps of the process, the methods, compositions and kits provided hereinimprove the 3C process and overcome the above-mentioned obstacles.Furthermore, the methods, compositions and kits disclosed herein resultin an increase in library yield and a several fold increase in ligationefficiency. The present teachings also accommodate a wide range of cellinput in a much smaller reaction volume. The present teachings alsoprovide validated TaqMan® control assays that allow for the quantitativemeasurement and control of digestion and ligation efficiencies. Themethods, compositions and kits provided herein also are useful forhigher-order genome analysis.

In addition, the 3C analysis methods currently available are not capableof high throughput analysis and therefore can only analyze a limitednumber of loci. Other methods for three-dimensional analysis ofchromatin structure are limited in their ability to capture and identifylong-range interactions and are biased towards proximal genomicinteractions.

The methods, compositions and kits disclosed herein overcome these andother limitations by connecting DNA fragments cross-linked throughprotein complexes with short non-palindromic DNA half-adaptors. Theresulting library is estimated to include millions of DNA molecules inwhich the ligation of two half-adaptors connects two DNA fragments. Theresulting full adaptor (ligation of two complementary half-adaptors) maythen serve as a primer template to analyze the library by DNA sequencingto identify potentially interacting genomic loci joined at each end ofthe full adaptor. Alternatively, the library may be sequenced from bothends to identify the interacting loci. In addition, the DNA fragmentslinked by an adaptor molecule may be analyzed by the polymerase chainreaction (PCR). The methods, compositions and kits of the presentteachings may be used to capture the interaction of all or a significantfraction of genomic elements that are proximal and long-range andcross-linked in vivo. This approach allows for the detection ofgenomic-wide binary contact in a high-throughput and unbiased manner.

Aspects of the present teachings may be further understood in light ofthe following detailed description and examples, which should not beconstrued as limiting the scope of the present teachings in any way. Thesection headings used herein are for organizational purposes only andare not to be construed as limiting the described subject matter in anyway. All literature and similar materials cited in this specification,including, but not limited to, patents, patent applications, articles,books, treatises and Internet web pages are expressly incorporated byreference in their entirety for any purpose. When definitions of termsin incorporated references appear to differ from the definitionsprovided in the present teachings, the definition provided in thepresent teachings shall control. It will be appreciated that there is animplied “about” prior to the temperatures, concentrations, times, etc.discussed in the present teaching, such that slight and insubstantialdeviations are within the scope of the present teachings herein. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. For example, “a primer” means that morethan one primer can, but need not, be present; for example, but withoutlimitation, one or more copies of a particular primer species, as wellas one or more versions of a particular primer type, for example but notlimited to, a multiplicity of different forward primers. Also, the useof “comprise,” “comprises,” “comprising,” “contain,” “contains,”“containing,” “include,” “includes,” and “including” are not intended tobe limiting. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive of the invention of the presentteachings.

A comparison of the methods disclosed herein to the 3C method of Dekkeret al., (supra) reveals a shorter, more streamlined process resulting inincreased efficiency. The 3C method of Dekker et al. is a five-dayprocess whereas the methods disclosed herein require only two days toobtain a result (see, Table 1 below).

TABLE 1 Comparison of Chromosomal Conformation Analysis Methods ProtocolDay 1 Day 2 Day 3 Day 4 Day 5 3C (Dekker) Cross-linking Restriction Pool& transfer to Spin bottles at high Template Cell lysis enzyme 3-50 mltubes speed, 20 min input Homogenization inactivation Phenol extraction(4° C.) titration Split into 20 Change to 8- Phenol: ChloroformResuspend in 500 PCR setup tubes 15 ml tubes 1:1 extraction μl Tris/EDTAPCR Wash in Ligation Pool & transfer to buffer Separate on restrictionbuffer (16° C., 2 h) 250 ml bottles Phenol extraction 1.5% 3× ProteinaseK Ethanol precipitate Phenol: Chloroform agarose gel Restriction (65°C., 2 h, (-80° C. overnight) 1:1 extraction Gel enzyme then Ethanolprecipitate imaging & digestion overnight) Wash in 70% band overnight(37° C.) Phenol plus ethanol, 7× quantitation buffer overnightEmbodiments Cross-linking Purification N/A N/A N/A of the Cell lysiswith beads methods Wash in Template described restriction buffer qualityherein 3× check Restriction enzyme digestion (37° C., 2 h) Restrictionenzyme inactivation Ligation (16° C., 60 min) Proteinase K (65° C.,overnight)

One embodiment provides a method of chromosome conformation analysis(see, FIG. 1), the method including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction endonuclease;

e) ligating the digested DNA by incubating with a ligating agent therebycreating a ligation product;

f) reversing the cross-linking;

g) purifying the ligated DNA; and h) analyzing the ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment of the methods disclosed herein, cross-linking the DNAand the proteins interacting with the DNA is performed by using across-linking reagent, such as an agent that can reversibly cross-linkprimary amino groups in proteins with other nearby nitrogen atoms inother proteins or DNA. Such cross-linking agents include, but are notlimited to, formaldehyde, paraformaldehyde, formalin, other similaraldehyde compounds, and other bi-functional cross-linking reagents thatcan covalently cross-link protein-protein and protein-DNA moleculestogether. The cross-linking reaction may be quenched, or inhibited, bythe addition of an amine-containing quenching reagent. Exemplaryamine-containing quenching reagents include, any compound that containsan available amine group including, but not limited to, methylamine,ethanolamine, Tris, dimethylamine, methylethanolamine, trimethylamine,aziridine, piperidiene, amiline, glycine, asparagine, and glutamine.

In the methods disclosed herein, the cross-linked DNA may be lysed in adetergent-containing solution. The detergents that may be used inembodiments of the methods disclosed herein include, but are not limitedto, anionic detergents, cationic detergents, non-ionic detergents andzwitterionic detergents, or combinations thereof. Such combinationsinclude: one or more cationic detergents with one or more anionicdetergents; one or more cationic detergents with one or more non-ionicdetergents; one or more cationic detergents with one or morezwitterionic detergents; one or more anionic detergents with one or morenon-ionic detergents; one or more anionic detergents with one or morezwitterionic detergents; and one or more non-ionic detergents with oneor more zwitterionic detergents. In a preferred embodiment, one or moreanionic detergents are combined with one or more non-ionic detergents.Non-limiting examples of anionic detergents include alkyl sulfates(e.g., sodium dodecylsulfate), alkyl sulfonates (e.g., octane sulfonicacid), bile salts, docusate sodium salt, and N-laurylsarcosine.Non-limiting examples of cationic detergents include bezalkoniumchloride, cetyl pyridium chloride, dodecyltrimethylammonium chloride,Girard's reagent, and Hyamine® 1622. Non-limiting examples of non-ionicdetergents include Tween®-20, Tween®-80, Triton® X-100, Triton® X-114,PEGylates, IGEPAL, Nonidet™-P40, Pluronic® F-68, Poloxamer 407, saponinand Tergitol®. Non-limiting examples of zwitterionic detergents includeCHAPS, L-α-lysophosphatidyl choline, DDMAB, and miltefosine.

After the lysis step, the cross-linked DNA may be digested usingrestriction endonucleases. As used herein, the terms “restrictionendonuclease” and “restriction enzyme” are equivalent and are usedinterchangeably. Any type of restriction endonuclease may be used todigest the cross-linked DNA. Preferably, the restriction endonucleasehas a six base recognition sequence, but it may have an eight baserecognition sequence, a seven base recognition sequence, a five baserecognition sequence or a four base recognition sequence. Suchrestriction endonucleases that may be used in the methods describedherein include, but are not limited to, Alu I, Apo I, Ase I, BamH I,BfuC I, Bgl II, BsaJ I, BstKT I, BstY I, Btg I, Cla I, CviKI-1, Dpn I,Dpn II, Eco47 III, EcoR I, EcoR V, EcoP15I, Fai I, Hae III, Hind III,Hpa II, HpyCH4 III, Kpn I, Mbo I, Mnl I, Mse I, Msp I, Nco I, Nde I, NheI, Not I, Pst I, Rsa I, Sac I, Sac II, Sal I, Sau3A I, Sfi I, Sma I, TaqI, Tsp509 I, Xba I, Xho I and Xma I. In a preferred embodiment, therestriction enzyme is EcoRI and HindIII. The restriction endonucleasedigestion may be terminated by the addition of a restriction stopsolution and a neutralizing solution. In another embodiment, therestriction endonuclease digestion may be terminated by heat treatment.

After the digestion step, the cross-linked DNA fragments may be ligatedusing a ligase that is specific for double-stranded DNA. Such ligasesinclude, but are not limited to, T4 DNA ligase, Tfi DNA ligase, DNAligase I, DNA ligase II, DNA ligase III, DNA ligase IV, and smallfootprint DNA ligases.

Once the ligation step is completed, the cross-linking is reversed usingfor example, a protease such as, but not limited to, serine proteases,such as Proteinase K, chymotrypsin, trypsin, elastin, subtilisin;threonine proteases; cysteine proteases, such as caspase, cathepsins,papain; aspartate proteases, such as rennin, chymosin, cathepsin D,pepsin; metalloproteases, such as aminopeptidase, dipeptidylpeptidase,angiotensin converting enzyme, carboxypeptidase; and glutamic acidpeptidases. In preferred embodiments, the protease is Proteinase K.Following reversal of cross-linking, the ligated DNA is purified.

In one embodiment, the purifying step includes purifying the ligationproduct using column purification. In another embodiment, the purifyingstep includes incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product may be collected either bycentrifugation or addition of a magnetic field. The beads aresubsequently washed and the ligated DNA is eluted.

The use of magnetic beads replaces the phenol/chloroform DNAprecipitation steps and advantageously results in a higher yield ofcross-linked product due to the omission of the multiple extraction anddilution steps. In addition, incubation with the magnetic beads replacesthe need for the lengthy phenol:chloroform extraction and results inavoiding the use of toxic chemicals.

Another embodiment provides a chromosome conformation analysis methodthat includes a control assay that monitors the undigested template DNA,herein denoted “Assay 1.” (see, FIG. 2) Assay 1 uses oligonucleotideprimers that specifically hybridize to regions either upstream (hereindenoted “forward primer”) or downstream (herein denoted “reverseprimer”) of the restriction endonuclease cutting site in the templateDNA. If the restriction site is not digested, the sequence will beamplified. An oligonucleotide probe that hybridizes to a sequenceupstream of the restriction site may be used to monitor the presence ofthe undigested template DNA. In a preferred embodiment, the restrictionendonuclease recognizes a six-base cutting sequence. In a more preferredembodiment, the restriction endonuclease is selected from EcoRI andHindIII.

Yet another embodiment provides a chromosome conformation analysismethod that includes a control assay that monitors the digested templateDNA, herein denoted “Assay 2.” (see, FIG. 2) Assay 2 usesoligonucleotides, herein referred to as “bridge oligos” that have ablocked 3′ end, a template binding region that hybridizes to the 3′ endof the template DNA, and a 5′ region that hybridizes to the primer andprobe sequences. In the first cycle of PCR, the bridge oligo anneals tothe template DNA via the template DNA binding region and the bridgeoligo sequence is amplified resulting in a first amplification product.In the second cycle of PCR, first amplification product is incubatedwith a forward primer that hybridizes to the 5′ end of the bridge oligothat contains the primer binding site which is upstream of therestriction endonuclease cutting site, and a reverse primer thathybridizes to the template DNA which contains the complement of thebridge oligo, and corresponds to a region downstream of the restrictionendonuclease cutting site, to amplify the bridge oligo if the templateDNA is digested. An oligonucleotide probe that hybridizes to a sequencethat contains the restriction endonuclease cutting site, which iscontained within the bridge oligo, may be used to monitor the presenceof the digested DNA. In a preferred embodiment, the restrictionendonuclease recognizes a six-base cutting sequence. In a preferredembodiment, the restriction endonuclease is selected from EcoRI andHindIII.

Another embodiment of the chromosomal conformation methods disclosedherein provides a method for analyzing non-specific between-cellligation (see, FIG. 3), the method including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) dividing the digested DNA into two separate aliquots resulting in afirst aliquot and a second aliquot;

g) ligating a first half-adaptor to the digested DNA of the firstaliquot thereby forming a first ligation product and a secondhalf-adaptor to the digested DNA of the second aliquot thereby forming asecond ligation product, wherein the first and second half-adaptors aredifferent from each other and each half-adaptor contains anon-palindromic overhang on one end and a T overhang on the other end,wherein the non-palindromic overhang of the first half-adaptor iscomplementary to the non-palindromic overhang of the secondhalf-adaptor;

h) combining the first and second ligation products;

i) ligating the first and second ligation products thereby forming athird ligation product; and

j) detecting and/or quantitating the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is then eluted from thebeads.

In one embodiment of the methods disclosed herein, half-adaptors areprovided. As provided herein, half-adaptors are double-stranded DNAmolecules that have a T overhang on one end and a non-palindromic orcohesive overhang on the other end (FIG. 3). A full adaptor is formed inthe following manner: half-adaptors are ligated to free ends of the DNAgenerated by restriction digestion of the cross-linked DNA through T:Aligation and ligation of two half-adaptors joined by their cohesive(e.g., non-palindromic) overhangs.

Another embodiment provides a method for determining thethree-dimensional arrangement of chromatin in a cell (see, FIG. 4), themethod including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) ligating a first half-adaptor to the digested DNA thereby forming afirst ligation product and a second half-adaptor to the digested DNAthereby forming a second ligation product, wherein the first and secondhalf-adaptors are different from each other and each half-adaptorcomprises a non-palindromic overhang on one end and a T overhang on theother end, wherein the non-palindromic overhang of the first adaptor iscomplementary to the non-palindromic overhang of the second adaptor;

g) phosphorylating the first and second ligation products;

h) ligating the first and second ligation products to form a thirdligation product; and

i) analyzing the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors have a stem-loopstructure. In yet another embodiment, one of the first and secondhalf-adaptors is linear and the other has a stem-loop structure. In yeta further embodiment, one of the first and second half-adaptors isconjugated to biotin.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is then eluted from thebeads.

Yet another embodiment (see, FIG. 5) provides a chromosome conformationmethod including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) incubating the digested DNA with a phosphatase;

f) ligating a first looped half-adaptor to the digested DNA therebyforming a first ligation product and a second looped half-adaptor to thedigested DNA thereby forming a second ligation product, wherein thefirst and second looped half-adaptors are different from each other andeach looped half-adaptor comprises a stem-loop structure, wherein the 5′end of the stem comprises a cohesive (i.e., non-palindromic) end thatterminates in a phosphate group;

g) heating the first and second ligation products thereby allowing thecohesive ends of each looped half-adaptors to anneal to each other;

h) phosphorylating the free ends of each of the looped half-adaptors;

i) ligating the looped half-adaptors to the DNA fragment thereby forminga third ligation product; and

i) analyzing the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase. In one embodiment, the heating andannealing step is replaced by a nick translation step.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors have a stem-loopstructure. In yet another embodiment, one of the first and secondhalf-adaptors is linear and the other has a stem-loop structure. In yeta further embodiment, one of the first and second half-adaptors isconjugated to biotin.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is then eluted from thebeads.

Yet a further embodiment (see, FIG. 6) provides a chromosomalconformation method including the steps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) ligating a first looped half-adaptor to the digested DNA therebyforming a first ligation product and a second looped half-adaptor to thedigested DNA thereby forming a second ligation product, wherein thefirst and second looped half-adaptors are different from each other andeach looped half-adaptor comprises a stem-loop structure, wherein the 5′end of the stem comprises an —OH group and a 3′ T overhang;

g) heating the first and second ligation products thereby allowing eachof the looped half-adaptors to anneal to each other;

h) phosphorylating the free ends of each of the looped half-adaptors;

i) ligating the half-adaptors to the DNA fragment thereby forming athird ligation product; and

i) analyzing the third ligation product.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme recognizes a six-base cuttingsequence. In a preferred embodiment, the restriction enzyme is selectedfrom EcoRI and HindIII. In another embodiment, the restriction enzymeyields a blunt end. In another embodiment, the filling-in step usesKlenow (exo−) DNA polymerase. In one embodiment, the filling-in step isreplaced by a phosphatase step. In another embodiment thephosphorylation and ligation steps are replaced by a nick translationstep.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors have a stem-loopstructure. In yet another embodiment, one of the first and secondhalf-adaptors is linear and the other has a stem-loop structure. In yeta further embodiment, one of the first and second half-adaptors isconjugated to biotin.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is then eluted from thebeads.

Another embodiment provides a method of creating a library forchromosome conformation analysis (see, FIG. 7), the method including thesteps of:

a) isolating cells from a biological sample;

b) incubating the cells with a cross-linking agent to cross-linkproteins and DNA (e.g., chromatin or gDNA), thereby forming across-linked product;

c) lysing the cells;

d) digesting the DNA with a restriction enzyme;

e) filling-in and A-tailing the free ends of the digested DNA;

f) optionally methylating the digested DNA;

g) ligating a first half-adaptor to the digested DNA thereby forming afirst ligation product and a second half-adaptor to the digested DNAthereby forming a second ligation product, wherein the first and secondhalf-adaptors are different from each other and each half-adaptorcomprises a non-palindromic overhang on one end and a T overhang on theother end, wherein the non-palindromic overhang of the first adaptor iscomplementary to the non-palindromic overhang of the second adaptor,further wherein the first or the second half-adaptor is biotinylated;

h) phosphorylating the ligated half-adaptors;

i) nick ligating the first and second ligation products, thereby forminga third ligation product;

j) reversing the cross-linking;

k) purifying the third ligation product;

l) digesting the third ligation product with a restriction endonuclease;

m) filling-in and A-tailing the free ends of the digested DNA

n) isolating the digested DNA using streptavidin beads;

o) ligating the digested DNA with sequencing primers;

p) analyzing the DNA.

In one embodiment, the cross-linked product is optionally incubated witha cross-linking quencher. In another embodiment, the cross-linkedproduct is removed from the cross-linking agent by washing or separatingthe quencher from the cross-linked product. In one embodiment, the cellsare in suspension. In another embodiment, the cells are adherent. Thecells may be intact, live, permeabilized or otherwise treated dependingon the method of isolation.

In one embodiment, the ligation products are analyzed by the polymerasechain reaction (PCR). In preferred embodiments, the PCR can be endpointPCR, real-time PCR, qPCR and dPCR, most preferably, the PCR is real-timePCR and qPCR. In one embodiment, the ligation products are analyzed byDNA sequencing methods including, but not limited to, fragment analysis,dideoxy sequencing (i.e., Sanger sequencing), Maxam-Gilbert chaintermination sequencing, dye terminator sequencing, dye primersequencing, pyrosequencing, next generation sequencing methodsincluding, high-throughput sequencing, including massively parallelsignature sequencing, SOLiD sequencing (e.g., sequencing by ligation),sequencing by hybridization, proton ion semiconductor sequencing, DNAnanoball sequencing, single molecule sequencing, and nanoporesequencing.

In one embodiment, the restriction enzyme is selected from a Type IIS orType III restriction endonuclease. In a preferred embodiment, therestriction enzyme is selected from MmeI and EcoP15I. In anotherembodiment, the restriction enzyme yields a blunt end. In anotherembodiment, the filling-in step uses Klenow (exo−) DNA polymerase.

In one embodiment, the first and second half-adaptors are linear. Inanother embodiment, the first and second half-adaptors have a stem-loopstructure. In yet another embodiment, one of the first and secondhalf-adaptors is linear and the other has a stem-loop structure.

In another embodiment, the third ligation product is purified before theanalysis and/or detection step. In one embodiment, the purifying stepcomprises incubating the ligation product with magnetic beads for aperiod of time sufficient to allow the ligation product to bind to themagnetic beads. The bound ligation product is then eluted from thebeads.

In one embodiment of the methods disclosed herein, cross-linking the DNAand the proteins interacting with the DNA is performed by using across-linking reagent, such as an agent that can reversibly cross-linkprimary amino groups in proteins with other nearby nitrogen atoms inother proteins or DNA. Such cross-linking agents include, but are notlimited to, formaldehyde, paraformaldehyde, formalin, other similaraldehyde compounds, and other bi-functional cross-linking reagents thatcan covalently cross-link protein-protein and protein-DNA moleculestogether. The cross-linking reaction may be quenched, or inhibited, bythe addition of an amine quenching reagent. Exemplary amine quenchingreagents include, but are not limited to, methylamine, ethanolamine,Tris, dimethylamine, methylethanolamine, trimethylamine, aziridine,piperidiene, amiline, glycine, asparagine, and glutamine.

In the methods disclosed herein, the cross-linked cells and DNAcomplexes may be lysed. In preferred embodiments, the cells are lysed ina detergent-containing solution. The detergents that may be used inembodiments of the methods disclosed herein include, but are not limitedto, anionic detergents, cationic detergents, non-ionic detergents andzwitterionic detergents, or combinations thereof. Such combinationsinclude: one or more cationic detergents with one or more anionicdetergents; one or more cationic detergents with one or more non-ionicdetergents; one or more cationic detergents with one or morezwitterionic detergents; one or more anionic detergents with one or morenon-ionic detergents; one or more anionic detergents with one or morezwitterionic detergents; and one or more non-ionic detergents with oneor more zwitterionic detergents. Preferred combinations are one or moreionic detergent with one or more non-ionic detergent. Non-limitingexamples of anionic detergents include alkyl sulfates (e.g., sodiumdodecylsulfate), alkyl sulfonates (e.g., octane sulfonic acid), bilesalts, docusate sodium salt, and N-laurylsarcosine. Non-limitingexamples of cationic detergents include bezalkonium chloride, cetylpyridium chloride, dodecyltrimethylammonium chloride, Girard's reagent,and Hyamine® 1622. Non-limiting examples of non-ionic detergents includeTween®-20, Tween® 80, Triton® X-100, Triton® X-114, PEGylates, IGEPAL,Nonidet™-P40, Pluronic® F-68, Poloxamer 407, saponin and Tergitol®.Non-limiting examples of zwitterionic detergents include CHAPS,L-α-lysophosphatidyl choline, DDMAB, and miltefosine.

After the lysis step, the cross-linked DNA may be digested. In preferredembodiments, the DNA is digested using restriction endonucleases. Anytype of restriction endonuclease may be used to digest the cross-linkedDNA. Preferably, the restriction endonuclease has a six base recognitionsequence, but it may have an eight base recognition sequence, a sevenbase recognition sequence, a five base recognition sequence or a fourbase recognition sequence. Such restriction endonucleases that may beused in the methods described herein include, but are not limited to,Alu I, Apo I, Ase I, BamH I, BfuC I, Bgl II, BsaJ I, BstKT I, BstY I,Btg I, Cla I, CviKI-1, Dpn I, Dpn II, Eco47 III, EcoR I, EcoR V,EcoP15I, Fai I, Hae III, Hind III, Hpa II, HpyCH4 III, Kpn I, Mbo I, MnlI, Mse I, Msp I, Nco I, Nde I, Nhe I, Not I, Pst I, Rsa I, Sac I, SacII, Sal I, Sau3A I, Sfi I, Sma I, Taq I, Tsp509 I, Xba I, Xho I and XmaI. After digestion, the restriction endonuclease digestion may beterminated. In preferred embodiments, the digestion may be terminated bythe addition of a restriction stop solution and a neutralizing solution.In another embodiment, the endonuclease digestion may be stopped by heattreatment.

After the digestion step, the free ends of the digested DNA fragmentsthat have a 5′-overhang may be filled in with a dNTP. In preferredembodiments, the overhang is filled-in using Klenow enzyme (exo⁻). Byadding the dA moiety to the free ends of the digested DNA fragments,ligation to the half-adaptors with a T overhang is facilitated.

In one embodiment, each half-adaptor is a double-stranded DNA moleculethat has a T overhang on one end and a non-palindromic or cohesiveoverhang on the other end (see FIGS. 3-7). A full adaptor can begenerated in the following manner: half-adaptors are ligated to free DNAends generated by restriction digestion of cross-linked chromatinthrough T:A ligation and by ligating two half-adaptors joined by theircohesive (i.e., non-palindromic) overhangs. Other methods of ligation,including blunt-ended ligation and cohesive overhang ligation may beused depending on the restriction enzyme used.

In another embodiment, each half-adaptor is a double-stranded DNAmolecule that comprises a stem-loop conformation (herein referred to asa “looped adaptor”), wherein the 5′ end of the stem comprises a cohesive(or non-palindromic) overhang that terminates in a 5′ phosphate group.Furthermore, the overhang portion of each looped half-adaptor iscomplementary to the other (see FIG. 5). In this embodiment, a fulladaptor is formed by the overhang portions of the two loopedhalf-adaptors annealing with each other (e.g., the overhang portion oflooped half-adaptor “A” is complementary to and anneals with theoverhang portion of looped half-adaptor “B”).

In another embodiment, each half-adaptor is a looped half-adaptor,wherein the 5′ end of the stem comprises a 5′-OH group and a 3′T-overhang. In addition, the overhang portion of each loopedhalf-adaptor is complementary to the other (see FIG. 6). In thisembodiment, a full adaptor is formed by the overhang portions of the twolooped half-adaptors annealing with each other (e.g., the overhangportion of looped half-adaptor “A” is complementary to and anneals withthe overhang portion of looped half-adaptor “B”).

The use of half-adaptors with non-palindromic overhangs removes the biasfor ligation of proximal sites and allows for increased ligation oflong-range interacting sites because each free end of the digested DNAcan ligate to either of the two types of half-adaptors. For example,each free end of the digested DNA may be ligated to half-adaptor “A” orhalf-adaptor “B”; however, only half-adaptors A and B will ligate witheach other to form a full adaptor. The other combinations ofhalf-adaptors, e.g., A-A and B-B will not form a full adaptor.Therefore, only 50% of the possible half-adaptor ligation events willresult in the formation of a full adaptor. Accordingly, if the mostproximal half-adaptor is not capable of forming a full adaptor, thehalf-adaptor will ligate with the next available half-adaptor that willform a full adaptor.

After digestion and dA tailing, the cross-linked DNA fragments may beligated using a ligase that is specific for double-stranded DNA. Suchligases include, but are not limited to, T4 DNA ligase, Tfi DNA ligase,DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV, and smallfootprint DNA ligases.

In addition, depending on the type of half-adaptors used, the adaptorsmay be phosphorylated in order to facilitate the ligation. Kinasesuseful for phosphorylating polynucleotides include, but are not limitedto, T7 polynucleotide kinase and T4 polynucleotide kinase (PNK).Preferably, the kinase is T4 polynucletoide kinase.

In other embodiments of the present teachings, nick-translation with,for example, E. coli DNA polymerase I, is carried out instead of thephosphorylation and ligation of annealed looped adaptors. In nicktranslation, free 3′-hydroxyl groups are created within the DNA by“nicks” caused by DNase I treatment or by the free 3′-OH groupsresulting from the annealed looped adaptors. DNA polymerase I thencatalyzes the addition of a nucleotide (labeled or unlabeled) to the3′-hydroxyl terminus of the nick. At the same time, the 5′-to-3′exonuclease activity of this enzyme eliminates the nucleotide unit fromthe 5′-phosphoryl terminus of the nick. A new nucleotide with a free3′-OH group is incorporated at the position of the original excisednucleotide unit in the 3′ direction.

Once ligation of the digested genomic DNA with half-adaptors to create afull adaptor is completed, the cross-linking is reversed, for examplewith Proteinase K, and the ligated DNA is purified. Purification may beperformed using several methods known in the art including, but notlimited to, magnetic beads, spin columns, gel purification, and affinitypurification using, for example, biotin/streptavidin.

As used herein, the term “DNA” refers to deoxyribonucleic acid in itsvarious forms as understood in the art, such as genomic DNA (gDNA),cDNA, isolated nucleic acid molecules, vector DNA, chromosomal DNA andchromatin. “Nucleic acid” refers to DNA or RNA in any form.

As used herein, the term “enzymatically active mutant or variantthereof,” when used in reference to an enzyme such as a polymerase or aligase, means a protein with appropriate enzymatic activity. Thus, forexample, but without limitation, an enzymatically active mutant orvariant of a DNA polymerase is a protein that is able to catalyze thestepwise addition of appropriate deoxynucleoside triphosphates into anascent DNA strand in a template-dependent manner. An enzymaticallyactive mutant or variant differs from the “generally-accepted” orconsensus sequence for that enzyme by at least one amino acid,including, but not limited to, substitutions of one or more amino acids,addition of one or more amino acids, deletion of one or more aminoacids, and alterations to the amino acids themselves. With the change,however, at least some catalytic activity is retained. In certainembodiments, the changes involve conservative amino acid substitutions.Conservative amino acid substitution may involve replacing one aminoacid with another that has, for example, similar hydrophobicity,hydrophilicity, charge, or aromaticity. In certain embodiments,conservative amino acid substitutions may be made on the basis ofsimilar hydropathic indices. A hydropathic index takes into account thehydrophobicity and charge characteristics of an amino acid, and incertain embodiments, may be used as a guide for selecting conservativeamino acid substitutions. It is understood in the art that conservativeamino acid substitutions may be made on the basis of any of theaforementioned characteristics.

Alterations to the amino acids may include, but are not limited to,glycosylation, methylation, phosphorylation, biotinylation, and anycovalent and noncovalent additions to a protein that do not result in achange in amino acid sequence. “Amino acid” as used herein refers to anyamino acid, natural or nonnatural, that may be incorporated, eitherenzymatically or synthetically, into a polypeptide or protein.

Fragments, for example, but without limitation, proteolytic cleavageproducts, are also encompassed by this term, provided that at least someenzyme catalytic activity is retained.

The skilled artisan will readily be able to measure catalytic activityusing an appropriate well-known assay. Thus, an appropriate assay forpolymerase catalytic activity might include, for example, measuring theability of a variant to incorporate, under appropriate conditions, rNTPsor dNTPs into a nascent polynucleotide strand in a template-dependentmanner. Likewise, an appropriate assay for ligase catalytic activitymight include, for example, the ability to ligate adjacently hybridizedoligonucleotides comprising appropriate reactive groups.

The term “sample” refers to any substance comprising nucleic acidmaterial.

As used herein, the term “probe” comprises a polynucleotide thatcomprises a specific portion designed to hybridize in asequence-specific manner with a complementary region of a specificnucleic acid sequence, e.g., a target nucleic acid sequence. In certainembodiments, the specific portion of the probe may be specific for aparticular sequence, or alternatively, may be degenerate, e.g., specificfor a set of sequences. In certain embodiments, the probe is labeled.

As used herein, the term “hybridization” refers to the complementarybase-pairing interaction of one nucleic acid with another nucleic acidthat results in the formation of a duplex, triplex or otherhigher-ordered structure.

As used herein, the term “anneal” refers to specific interactionsbetween strands of nucleotides wherein the strands bind to one anothersubstantially based on complementarity between the strands as determinedby Watson-Crick base pairing or Hoogstein-type hydrogen bonding.Base-stacking and hydrophobic interactions may also contribute to duplexstability. Conditions for hybridizing probes and primers tocomplementary and substantially complementary target sequences are wellknown. In general, whether such annealing takes place is influenced by,among other things, the length of the polynucleotides and thecomplementarity between the bases, the pH, the temperature, the presenceof mono- and divalent cations, the proportion of G and C nucleotides inthe hybridizing region, the viscosity of the medium, and the presence ofdenaturants. Such variables influence the time required forhybridization. Thus, the preferred annealing conditions will depend uponthe particular application. Such conditions, however, may be routinelydetermined by the person of ordinary skill in the art without undueexperimentation. It will be appreciated that complementarity need not beperfect; there can be a small number of base pair mismatches that willminimally interfere with hybridization between the target sequence andthe single-stranded nucleic acids of the present teachings. However, ifthe number of base pair mismatches is so great that no hybridization canoccur under minimally stringent conditions, then the sequence isgenerally not a complementary target sequence. Thus, “complementarity”herein is meant that the probes or primers are sufficientlycomplementary to the target sequence to hybridize under the selectedreaction conditions to achieve the ends of the present teachings.

As used herein, the term “amplifying” refers to any means by which atleast a part of a nucleotide sequence, target polynucleotide, targetpolynucleotide surrogate, or combinations thereof, is reproduced,typically in a template-dependent manner, including without limitation,a broad range of techniques for amplifying nucleic acid sequences,either linearly or exponentially. Any of several methods may be used toamplify the target polynucleotide. These include linear, logarithmic, orany other amplification method. Exemplary methods include polymerasechain reaction (PCR; see, e.g., U.S. Pat. Nos. 4,683,202; 4,683,195;4,965,188; and 5,035,996), isothermal procedures (using one or more RNApolymerases (see, e.g., WO 2006/081222), strand displacement (see, e.g.,U.S. Pat. No. RE39,007), partial destruction of primer molecules (see,e.g., WO 2006/087574)), ligase chain reaction (LCR) (see, e.g., Wu, etal. Genomics 4:560-569 (1990) and Barany, et al. Proc. Natl. Acad. Sci.USA 88:189-193 (1991)), Qβ RNA replicase systems (see, e.g., WO1994/016108), RNA transcription-based systems (e.g., TAS, 3SR), rollingcircle amplification (RCA) (see, e.g., U.S. Pat. No. 5,854,033; U.S.Pub. No. 2004/265897; Lizardi, et al. Nat. Genet. 19:225-232 (1998); andBailer, et al. Nucleic Acid Res. 26: 5073-5078 (1998)), and stranddisplacement amplification (SDA) (Little, et al. Clin. Chem. 45:777-784(1999)), among others. Many systems are suitable for use in amplifyingtarget nucleic acids and are contemplated herein as would be understoodby one of skill in the art.

In one embodiment, the amplification reaction is a 5′-nuclease assay(also commercially known as TaqMan®) performed using a nucleic acidpolymerase, such as DNA polymerase, at least one oligonucleotide primercapable of specifically hybridizing to a target polynucleotide (fromwhich the amplified target nucleic acid is amplified), at least onedetectable probe that hybridizes to the amplified target nucleic acid,and which may be incorporated into the at least one primer), and atleast one detectable nucleic acid binding agent (e.g., an intercalatingor non-intercalating dye) which may be introduced before, during orafter amplification. The probe typically contains a detectable labelcapable of emitting a signal that may be monitored to ascertain whetherthe target nucleic acid has been amplified. In some embodiments, theprobe is an oligonucleotide that hybridizes to the target nucleic acid3′ relative to the at least one primer. In some embodiments, thepolymerase has nuclease activity (i.e., 5′-to-3′ nuclease activity) forreleasing the probe from the amplified nucleic acid. In someembodiments, release from the amplified nucleic acid renders the probedetectable. In some embodiments, the probe comprises a detectable labeland a quencher molecule that quenches the detectable label when free butdoes not quench when the probe is hybridized to the amplified nucleicacid. In some embodiments, two or more probes may be used where at leastone probe has a detectable label and at least one other probe has aquencher molecule. When in sufficiently close proximity to one another,the quencher molecule typically suppresses the signal of the detectablelabel on the other probe. In some embodiments, two or more probes, eachhaving a different detectable label, may be used without quenchermolecules. In such embodiments, the probes are rendered detectable,either de novo or by exhibiting a different signal than either probealone, when in sufficiently close proximity to one another. Typically,the detectable label and quencher molecule are part of a single probe.As amplification proceeds, the polymerase digests the probe to separatethe detectable label from the quencher molecule. The detectable label(e.g., a fluorophore) is monitored during the reaction, where detectionof the label corresponds to the occurrence of nucleic acid amplification(i.e., the higher the signal the greater the amount of amplification).

Additional reagents, systems, or detectable labels that may be used inthe methods described herein include, for example, detectablelabel-quencher systems (e.g., FRET, salicylate/DTPA ligand systems (see,e.g., Oser, et al. Angew. Chem. Int. Ed. Engl. 29:1167-1169 (1990),displacement hybridization, homologous probes, assays described in EP070685), molecular beacons (e.g., NASBA), Scorpion® probes, lockednucleic acid (LNA) bases (Singh, et al. Chem. Commun. 4:455-456 (1998)),peptide nucleic acid (PNA) probes (Pellestor, et al. Eur. J. Hum. Gen.12:694-700 (2004)), Eclipse probes (Afonina, et al. Biotechniques32:940-949 (2002)), light-up probes (Svanvik, et al. Anal. Biochem.281:26-35 (2000)), molecular beacons (Tyagi, et al. Nat. Biotechnol.14:303-308 (1996)), tripartite molecular beacons (Nutiu, et al. NucleicAcids Res. 30:E94 (2002)), QuantiProbes (www.qiagen.com), HyBeacons®(French, et al. Mol. Cell. Probes 15:363-374 (2001)), displacementprobes (Li, et al. Nucleic Acids Res. 30:E5 (2002)), HybProbes(Cardullo, et al. Proc. Natl. Acad. Sci. USA 85:8790-8794 (1988)), MGBAlert (www.nanogen.com), Q-PNA (Fiandaca, et al. Genome Res. 11:609-613(2001)), Plexor® technology (www.Promega.com), LUX™ primers (Nazarenko,et al. Nucleic Acids Res. 30:E37 (2002)), Scorpion® primers (Whitcombe,et al. Nat. Biotechnol. 17:804-807 (1999)), AmpliFluor® (Sunrise)primers (Nazarenko, et al. Nucleic Acids Res. 25:2516-2521 (1997)),DzyNA primers (Todd, et al. Clin. Chem. 46:625-630 (2000)), and thelike. In each of these assays, the generation of amplification productsmay be monitored while the reaction is in progress. An apparatus fordetecting the signal generated by the detectable label may be used todetect, measure, and quantify the signal before, during, or afteramplification. The particular type of signal may dictate the choice ofdetection method. For example, in some embodiments, fluorescent dyes areused to label probes or amplified products. The probes bind tosingle-stranded or double-stranded amplified products, or the dyesintercalate into the double-stranded amplified products, andconsequently, the resulting fluorescence increases as the amount ofamplified product increases. The use of other methods or reagents isalso contemplated herein as would be understood by one of skill in theart.

As mentioned above, in some embodiments the detectable label may beattached to a probe which may be incorporated into a primer or mayotherwise bind to amplified target nucleic acid (for example, adetectable nucleic acid binding agent such as an intercalating ornon-intercalating dye). When using more than one detectable label, eachlabel should differ in its spectral properties such that the labels maybe distinguished from each other, or such that together the detectablelabels emit a signal that is not emitted by either detectable labelalone. Exemplary detectable labels include, but are not limited to, afluorescent dye or fluorophore (i.e., a chemical group that may beexcited by light to emit fluorescence or phosphorescence), “acceptordyes” capable of quenching a fluorescent signal from a fluorescent donordye, and the like.

Suitable detectable labels include, for example, fluorosceins (e.g.,5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5-FAM); 5-HAT(Hydroxy Tryptamine); 6-HAT; 6-JOE; 6-carboxyfluorescein (6-FAM); FITC);Alexa fluors (e.g., 350, 405, 430, 488, 500, 514, 532, 546, 555, 568,594, 610, 633, 635, 647, 660, 680, 700, 750); BODIPY® fluorophores(e.g., 492/515, 493/503, 500/510, 505/515, 530/550, 542/563, 558/568,564/570, 576/589, 581/591, 630/650-X, 650/665-X, 665/676, FL, FL ATP,FI-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-XSE), coumarins (e.g., 7-amino-4-methylcoumarin, AMC, AMCA, AMCA-S,AMCA-X, ABQ, CPM methylcoumarin, coumarin phalloidin, hydroxycoumarin,CMFDA, methoxycoumarin), calcein, calcein AM, calcein blue, calcium dyes(e.g., calcium crimson, calcium green, calcium orange, calcofluorwhite), Cascade Blue, Cascade Yellow; Cy™ dyes (e.g., 3, 3.18, 3.5, 5,5.18, 5.5, 7), cyan GFP, cyclic AMP Fluorosensor (FiCRhR), fluorescentproteins (e.g., green fluorescent protein (e.g., GFP. EGFP), bluefluorescent protein (e.g., BFP, EBFP, EBFP2, Azurite, mKalama1), cyanfluorescent protein (e.g., ECFP, Cerulean, CyPet), yellow fluorescentprotein (e.g., YFP, Citrine, Venus, YPet), FRET donor/acceptor pairs(e.g., fluorescein/tetramethylrhodamine, IAEDANS/fluorescein,EDANS/dabcyl, fluorescein/fluorescein, BODIPY® FL/BODIPY® FL,Fluorescein/QSY7 and QSY9), LysoTracker and LysoSensor (e.g.,LysoTracker Blue DND-22, LysoTracker Blue-White DPX, LysoTracker YellowHCK-123, LysoTracker Green DND-26, LysoTracker Red DND-99, LysoSensorBlue DND-167, LysoSensor Green DND-189, LysoSensor Green DND-153,LysoSensor Yellow/Blue DND-160, LysoSensor Yellow/Blue 10,000 MWdextran), Oregon Green (e.g., 488, 488-X, 500, 514); rhodamines (e.g.,110, 123, B, B 200, BB, BG, B extra, 5-carboxytetramethylrhodamine(5-TAMRA), 5 GLD, 6-Carboxyrhodamine 6G, Lissamine, Lissamine RhodamineB, Phallicidine, Phalloidine, Red, Rhod-2,5-ROX (carboxy-X-rhodamine),Sulphorhodamine B can C, Sulphorhodamine G Extra, Tetramethylrhodamine(TRITC), WT), Texas Red, Texas Red-X, VIC and other labels described in,e.g., US Pub. No. 2009/0197254), among others as would be known to thoseof skill in the art. Other detectable labels can also be used (see,e.g., US Pub. No. 2009/0197254), as would be known to those of skill inthe art.

As used herein, the term “incubating” refers to maintaining a state ofcontrolled conditions such as temperature over a period of time.

As used herein, the term “denaturation” refers to the separation ofnucleotide strands from an annealed state. Denaturation may be inducedby a number of factors including ionic strength of a buffer,temperature, or chemicals that disrupt base pairing interactions.

As used herein, the term “sufficient amount of time” when referring totime for an enzymatic reaction, refers to the time which allows theenzyme used to complete a reaction, such as amplification, ligation ordigestion. The amount of time required varies depending on severalfactors which are known in the art.

The term “or combinations thereof” as used herein refers to allpermutations and combinations of the listed items preceding the term.For example, “A, B, C or combinations thereof” is intended to include atleast one of: A, B, C, AB, AC, BC or ABC, and if order is important in aparticular context, also BA, CA, CB, CBA, BCA, ACB, BAC or CAB.Continuing with this example, expressly included are combinations thatcontain repeats of one or more item or term, such as BB, AAA, AAB, BBA,AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan willunderstand that typically there is no limit on the number of items orterms in any combination, unless otherwise apparent from the context.

As used herein, the term “reaction vessel” generally refers to anycontainer in which a reaction may occur in accordance with the presentteachings. In some embodiments, a reaction vessel may be amicrocentrifuge tube or other containers of the sort in common practicein modern molecular biology laboratories.

As used herein, the term “detection” refers to any of a variety of waysof determining the presence and/or quantity and/or identity of a targetpolynucleotide. In some embodiments employing a donor moiety and signalmoiety, one may use certain energy-transfer fluorescent dyes. Certainnonlimiting exemplary pairs of donors (donor moieties) and acceptors(signal moieties) are illustrated, e.g., in U.S. Pat. Nos. 5,863,727;5,800,996; and 5,945,526. Use of some combinations of a donor and anacceptor have been called FRET (Fluorescent Resonance Energy Transfer).In some embodiments, fluorophores that may be used as signaling probesinclude, but are not limited to, rhodamine, cyanine 3 (Cy3), cyanine 5(Cy5), fluorescein, VIC™, LIZ™, TAMRA™, 5-FAM™, 6-FAM™, and Texas Red(Molecular Probes, Eugene, Oreg.). (VIC™, LIZ™, TAMRA™, 5-FAM™, and6-FAM™ (all available from Life Technologies, Foster City, Calif.). Insome embodiments, the amount of detector probe that gives a fluorescentsignal in response to an excited light typically relates to the amountof nucleic acid produced in the amplification reaction. Thus, in someembodiments, the amount of fluorescent signal is related to the amountof product created in the amplification reaction. In such embodiments,one can therefore measure the amount of amplification product bymeasuring the intensity of the fluorescent signal from the fluorescentindicator.

Any quencher may be used without limitation in the methods andcompositions provided herein. The quencher may be located on either theprimer or the probe. Any quencher may be used as long as it decreasesthe fluorescence intensity of the fluorophore that is being used.Quenchers commonly used for FRET include, but are not limited to, DeepDark Quencher DDQ-I, DABCYL, Eclipse® Dark quencher, Iowa Black® FQ,BHQ-1, QSY-7, BHQ-2, DDQ-II, Iowa Black® RQ, QSY-21, and Black HoleQuencher® BHQ-3. Quenchers for use in the methods and compositionsprovided herein may be obtained commercially, for example, fromEurogentec (Belgium), Epoch Biosciences (Bothell, Wash.), BiosearchTechnologies (Novato Calif.), Integrated DNA Technologies (Coralville,Iowa) and Life Technologies (Carlsbad, Calif.).

According to some embodiments, one may employ an internal standard toquantify the amplification product indicated by the fluorescent signal.See, e.g., U.S. Pat. No. 5,736,333. Devices have been developed that mayperform a thermal cycling reaction with compositions containing afluorescent indicator, emit a light beam of a specified wavelength, readthe intensity of the fluorescent dye, and display the intensity offluorescence after each cycle. Devices comprising a thermal cycler,light beam emitter, and a fluorescent signal detector, have beendescribed, e.g., in U.S. Pat. Nos. 5,928,907; 6,015,674; and 6,174,670,and include, but are not limited to the ABI Prism® 7700 SequenceDetection System, the ABI GeneAmp® 5700 Sequence Detection System, theABI GeneAmp® 7300 Sequence Detection System, and the ABI GeneAmp® 7500Sequence Detection System (all available from Life Technologies, FosterCity, Calif.). In some embodiments, each of these functions may beperformed by separate devices. For example, if one employs a Q-betareplicase reaction for amplification, the reaction may not take place ina thermal cycler, but could include a light beam emitted at a specificwavelength, detection of the fluorescent signal, and calculation anddisplay of the amount of amplification product.

Any of the oligonucleotides provided herein (e.g., the primers or theprobes) may be DNA or RNA or chimeric mixtures or derivatives ormodified versions thereof, so long as the oligonucleotide is stillcapable of priming the desired polymerization reaction. Theoligonucleotide may be modified at the base moiety, sugar moiety, orphosphate backbone, and may include other appending groups or labels, solong as the substrate is still capable of priming the desiredpolymerization reaction.

For example, the oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide comprises at least onemodified sugar moiety selected from the group including, but not limitedto, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In some embodiments, the oligonucleotide comprises at least one modifiedphosphate backbone selected from the group including, but not limited toa phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In some embodiments, the oligonucleotides may be modified to morestrongly bind to their complementary oligonucleotide. Examples ofmodifications that may enhance the binding or an RNA or DNA or to itscomplementary oligonucleotide include, but are not limited to,2′-O-alkyl modified ribonucleotides, 2′-O-methyl ribonucleotides,2′-orthoester modifications (including but not limited to2′-bis(hydroxylethyl), and 2′ halogen modifications and locked nucleicacids (LNAs).

As used herein “polymerase” refers to any enzyme having a nucleotidepolymerizing activity. Polymerases (including DNA polymerases and RNApolymerases) useful in accordance with the present teachings include,but are not limited to, commercially available or natural DNA-directedDNA polymerases, DNA-directed RNA polymerases, RNA-directed DNApolymerases, and RNA-directed RNA polymerases. Polymerases used inaccordance with the invention may be any enzyme that can synthesize anucleic acid molecule from a nucleic acid template, typically in the 5′to 3′ direction.

Exemplary DNA polymerases that may be used in the methods, kits andcompositions provided herein include, but are not limited to: Thermusthermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™ DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillussterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNApolymerase, Sulfobus acidocaldarius (Sac) DNA polymerase, Thermoplasmaacidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNApolymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, mycobacterium DNA polymerase (Mtb, Mlep), and mutants,and variants and derivatives thereof. RNA polymerases such as T3, T5 andSP6 and mutants, variants and derivatives thereof may also be used inaccordance with the present teachings. Generally, any type I DNApolymerase may be used in accordance with the present teachings althoughother DNA polymerases may be used including, but not limited to, typeIII or family A, B, C etc., DNA polymerases.

The nucleic acid polymerases used in the methods, kits and compositionsprovided herein may be mesophilic or thermophilic. Exemplary mesophilicDNA polymerases include T7 DNA polymerase, T5 DNA polymerase, Klenowfragment DNA polymerase, DNA polymerase III and the like. Exemplarythermostable DNA polymerases include Taq, Tne, Tma, Pfu, Tfl, Tth,Stoffel fragment, VENT™ and DEEPVENT™ DNA polymerases, and mutants,variants and derivatives thereof (U.S. Pat. No. 5,436,149; U.S. Pat. No.4,889,818; U.S. Pat. No. 4,965,188; U.S. Pat. No. 5,079,352; U.S. Pat.No. 5,614,365; U.S. Pat. No. 5,374,553; U.S. Pat. No. 5,270,179; U.S.Pat. No. 5,047,342; U.S. Pat. No. 5,512,462; WO 92/06188; WO 92/06200;WO 96/10640; Barnes, W. M., Gene 112:29-35 (1992); Lawyer, F. C., etal., PCR Meth. Appl. 2:275-287 (1993); Flaman, J.-M, et al., Nucl. AcidsRes. 22(15):3259-3260 (1994)). Examples of DNA polymerases substantiallylacking in 3′ exonuclease activity include, but are not limited to, Taq,Tne (exo⁻), Tma (exo⁻), Pfu (exo⁻), Pwo (exo⁻) and Tth DNA polymerases,and mutants, variants and derivatives thereof.

DNA polymerases for use in the present teachings may be obtainedcommercially, for example, from Life Technologies, Inc. (Carlsbad,Calif.), Pharmacia (Piscataway, N.J.), Sigma (St. Louis, Mo.) andBoehringer Mannheim. Exemplary commercially available DNA polymerasesfor use in the present disclosure include, but are not limited to, TspDNA polymerase from Life Technologies, Inc.

In some embodiments, combined thermal cycling and fluorescence detectingdevices may be used for precise quantification of target nucleic acidsequences in samples. In some embodiments, fluorescent signals may bedetected and displayed during and/or after one or more thermal cycles,thus permitting monitoring of amplification products as the reactionsoccur in “real time.” In some embodiments, one may use the amount ofamplification product and number of amplification cycles to calculatehow much of the target nucleic acid sequence was present in the sampleprior to amplification. In some embodiments, one may simply monitor theamount of amplification product after a predetermined number of cyclessufficient to indicate the presence of the target nucleic acid sequencein the sample. One skilled in the art may easily determine, for anygiven sample type, primer sequence, and reaction condition, how manycycles are sufficient to determine the presence of a given targetpolynucleotide. As used herein, determining the presence of a target maycomprise identifying it, as well as optionally quantifying it. In someembodiments, the amplification products may be scored as positive ornegative as soon as a given number of cycles is complete. In someembodiments, the results may be transmitted electronically directly to adatabase and tabulated. Thus, in some embodiments, large numbers ofsamples may be processed and analyzed with less time and labor when suchan instrument is used.

In some embodiments, the ligation products may be analyzed by fragmentanalysis. In a preferred category of methods referred to herein as“fragment analysis” methods, labeled oligonucleotide fragments aregenerated through template-directed enzymatic synthesis using labeledprimers or nucleotides, e.g., by ligation or polymerase-directed primerextension; the fragments are subjected to a size-dependent separationprocess, e.g., electrophoresis or chromatography; and, the separatedfragments are detected subsequent to the separation, e.g., bylaser-induced fluorescence. In a preferred embodiment, multiple classesof oligonucleotides are separated simultaneously and the differentclasses are distinguished by spectrally resolvable labels.

One exemplary fragment analysis method is DNA sequencing. In general,DNA sequencing involves an extension/termination reaction of anoligonucleotide primer. Included in the reaction mixture aredeoxynucleoside triphosphates (dNTPs) which are used to extend theprimer. Also included in the reaction mixture is at least onedideoxynucleoside triphosphates (ddNTP) which when incorporated onto theextended primer prevents the further extension of the primer. After theextension reaction has been terminated, the different terminationproducts that are formed are separated and analyzed in order todetermine the positioning of the different nucleosides.

Fluorescent DNA sequencing may generally be divided into two categories:“dye primer sequencing” and “dye terminator sequencing.” In dye primersequencing, a fluorescent dye is incorporated onto the primer beingextended. Four separate extension/termination reactions are then run inparallel, each extension reaction containing a differentdideoxynucleoside triphosphates (ddNTP) to terminate the extensionreaction. After termination, the reaction products are separated by gelelectrophoresis and analyzed. See, for example, Ansorage et al., Nucl.Acids Res. 15:4593-4602 (1987).

In one variation of dye primer sequencing, different primers are used inthe four separate extension/termination reactions, each primercontaining a different spectrally resolvable dye. After termination, thereaction products from the four extension/termination reactions arepooled, electrophoretically separated, and detected in a single lane.See, for example, Smith et al., Nature 321:674-679 (1986). Thus, in thisvariation of dye primer sequencing, by using primers containing a set ofspectrally resolvable dyes, products from more than oneextension/termination reactions can be simultaneously detected.According to this method, a mixture of extended labeled primers areformed by hybridizing a nucleic acid sequence with a fluorescentlylabeled oligonucleotide primer in the presence of deoxynucleosidetriphosphates, at least one dideoxynucleoside triphosphates and a DNApolymerase. The fluorescently labeled oligonucleotide primer includes anoligonucleotide sequence complementary to a portion of the nucleic acidbeing sequenced, and a fluorescent dye attached to the oligonucleotide,preferably an energy transfer dye. According to this method, the DNApolymerase extends the primer with the deoxynucleoside triphosphatesuntil a dideoxynucleoside triphosphate is incorporated with terminatesextension of the primer. After termination, the mixture of extendedprimers is separated. The sequence of the nucleic acid sequence is thendetermined by fluorescently detecting the mixture of extended primersformed.

In dye terminator sequencing, a fluorescent dye is attached to each ofthe dideoxynucleoside triphosphates. An extension/termination reactionis then conducted where a primer is extended using deoxynucleosidetriphosphates until the labeled dideoxynucleoside triphosphates isincorporated onto the extended primer to prevent further extension ofthe primer. Once terminated, the reaction products for eachdideoxynucleoside triphosphates are separated and detected. In oneembodiment, separate extension/termination reactions are conducted foreach of the four dideoxynucleoside triphosphates. In another embodiment,a single extension/termination reaction is conducted which contains thefour dideoxynucleoside triphosphates, each labeled with a different,spectrally resolvable, fluorescent dye. According to this method, amixture of extended primers is formed by hybridizing a nucleic acidsequence with an oligonucleotide primer in the presence ofdeoxynucleoside triphosphates, at least one fluorescently labeleddideoxynucleotide triphosphates, and a DNA polymerase. The fluorescentlylabeled dideoxynucleotide triphosphates include a dideoxynucleosidetriphosphates labeled with a fluorescent dye, preferably an energytransfer dye. According to this method, the DNA polymerase extends theprimer with the deoxynucleoside triphosphates until a fluorescentlylabeled dideoxynucleoside triphosphates is incorporated onto theextended primer. After termination, the mixture of extended primers isseparated. The sequence of the nucleic acid sequence is then determinedby detecting the fluorescently labeled dideoxynucleoside attached to theextended primer.

In some embodiments, the sequence of at least part of the ligationproduct is determined thereby detecting the intra- and interchromosomalinteractions. The term “DNA sequencing” is used in a broad sense hereinand refers to any technique known in the art that allows the order of atleast some consecutive nucleotides in at least part of a DNA to beidentified. Some non-limiting examples of DNA sequencing techniquesinclude Sanger's dideoxy terminator method and the chemical cleavagemethod of Maxam and Gilbert, including variations of those methods;sequencing by hybridization, for example, but not limited to,hybridization of amplified products to a microarray or a bead, such as abead array; pyrosequencing, and restriction mapping. Some DNA sequencingmethods comprise electrophoresis, including without limitation,capillary electrophoresis and gel electrophoresis; mass spectroscopy;and single molecule detection. In some embodiments, DNA sequencingcomprises direct sequencing, duplex sequencing, cycle sequencing,single-base extension sequencing (SBE), solid-phase sequencing, orcombinations thereof. In some embodiments, DNA sequencing comprisesdetecting the sequencing product using an instrument, for example, butnot limited to, an ABI PRISM® 377 DNA Sequencer, an ABI PRISM® 310,3100, 3100-Avant, 3730 or 3730×1 Genetic Analyzer, an ABI PRISM® 3700DNA Analyzer or an Applied Biosystems SOLiD™ System, or an Ion PGM™sequencer (all available from Life Technologies, Carlsbad, Calif.), aGenome Sequencer 20 System (Roche Applied Science), or a massspectrometer. In certain embodiments, DNA sequencing comprises emulsionPCR (see, e.g., Williams et al, Nat. Methods 3:545-550 (2006)). Incertain embodiments, DNA sequencing comprises a high throughputsequencing technique, for example, but not limited to, massivelyparallel signature sequencing (MPSS). Descriptions of MPSS can be found,among other places, in Zhou et al, Methods of Molecular Biology,331:285-311, Humana Press Inc.; Reinartz et al, Briefings in FunctionalGenomics and Proteomics, 1:95-104 (2002); Jongeneel et al., GenomeResearch 15:1007-14 (2005)). In some embodiments, DNA sequencingcomprises incorporating a dNTP, including without limitation, a dATP, adCTP, a dGTP, a dTTP, a dUTP, a dITP, or combinations thereof, andincluding dideoxyribonucleotide versions of dNTPs, into an amplifiedproduct.

Further exemplary techniques that are useful for determining thesequence of at least a portion of a nucleic acid molecule include,without limitation, emulsion-based PCR followed by any suitablemassively parallel sequencing or other high-throughput technique. Insome embodiments, determining the sequence of at least a part of anamplified product to detect the corresponding RNA or DNA moleculecomprises quantitating the amplified product. In some embodiments,sequencing is carried out using the SOLiD™ System (Applied Biosystems)as described in, for example, PCT Application Publication No. WO06/084132 and WO 07/121,489. In some embodiments, sequencing is carriedout using the Ion PGM™ Sequencer, as discussed in, for example, Rothberget al., Nature 475:348-352 (2011). In some embodiments, quantitating theamplified product comprises real-time or end-point quantitative PCR orboth. In some embodiments, quantitating the amplified product comprisesone or more 5′-nuclease assays, for example, but not limited to, TaqMan®Gene Expression Assays, which may comprise a microfluidics deviceincluding without limitation, a low density array. Any suitableexpression profiling technique known in the art may be employed invarious embodiments of the disclosed methods.

Those in the art will appreciate that the sequencing method employed isnot typically a limitation of the present methods. Rather, anysequencing technique that provides the order of at least someconsecutive nucleotides of at least part of the corresponding amplifiedproduct or DNA to be detected may typically be used in the currentmethods. In some embodiments, unincorporated primers and/or dNTPs areremoved prior to a sequencing step by enzymatic degradation, includingwithout limitation, exonuclease I and shrimp alkaline phosphatasedigestion, for example, but not limited to, the ExoSAP-IT® reagent (USBCorporation). In some embodiments, unincorporated primers, dNTPs and/orddNTPs are removed by gel or column purification, sedimentation,filtration, beads, magnetic separation, or hybridization-based pull out,as appropriate.

In certain embodiments, the present teachings also provide kits designedto expedite performing certain methods. In some embodiments, kits serveto expedite the performance of the methods of interest by assembling twoor more components used in carrying out the methods. In someembodiments, kits may contain components in pre-measured unit amounts tominimize the need for measurements by end-users. In some embodiments,kits may include instructions for performing one or more methods of thepresent teachings. In certain embodiments, the kit components areoptimized to operate in conjunction with one another.

In another embodiment, the present compositions and methods may beassembled into kits for use in chromosome conformation analysis. Kitsaccording to this embodiment may comprise a carrier means, such as abox, carton, tube or the like, having in close confinement therein oneor more container means or reaction vessel, such as vials, tubes,ampoules, plates, bottles, and the like, wherein one or more containermeans contains: a cross-linking agent, a lysing solution, a DNA ligase,and a protease. In another embodiment, the kit further provides across-linking quencher. In yet another embodiment, the kit furtherprovides magnetic beads, a DNA binding solution, a DNA washing solution,and a DNA elution solution. In yet another embodiment, the kit furtherprovides a protease inhibitor cocktail, a restriction enzyme stopsolution, and a neutralization solution. In another embodiment, the kitcomprises control assays, wherein the control assays include Assay 1 andAssay 2, and a first and second half-adaptor. In a further embodiment,the kits disclosed herein may further comprise one or more of thefollowing: a buffer, such as phosphate buffer saline, and RNase A. In afurther embodiment, the kits disclosed herein may further compriseinstructions for carrying out the methods disclosed herein.

In a specific embodiment, the chromosome conformation kits may compriseone or more additional components, in mixtures or separately. Those ofskill in the art will understand that the additional components, eitherin the same tube or in separate tubes, may also be included in the kitto further facilitate or enhance reactions such as ligation,amplification, cross-linking and restriction digestion. Such componentsor additives, can include for example, Mg²⁺, uracil DNA glycosylase, apassive reference control to minimize sample-to-sample or well-to-wellvariations in quantitative real-time DNA-detection assays (e.g., dyessuch as ROX) and various hot start components (e.g., antibodies,oligonucleotides, beads, etc).

In another embodiment, the compositions in the present kits may beformulated as concentrated stock solutions (e.g., 2×, 3×, 4×, 5×, 6×,etc). In some embodiments, the compositions may be formulated asconcentrated stock solutions in a single tube or container.Collectively, some of the components of the kits according to thepresent teachings may be formulated together to create a master mix.Components of the kit other than the compositions disclosed herein maybe provided in individual containers or in a single container, asappropriate. Instructions and protocols for using the kit advantageouslymay be provided.

In some embodiments, the kit comprises a multi-well format platform,such as a 96-well plate, a 394-well plate, a 1536-well plate, an OpenArray™ multi-well plate, TaqMan® Low Density Array (TLDA) plate, an Ion314™ chip, an Ion 316™ chip, and an Ion 318™ chip. Such multi-wellformat platform may be used for analysis of multiple samples includingcontrol enzymes and multiple concentrations of the standard duplex forthe generation of a standard curve.

In another embodiment, compositions are provided. Typically, thecompositions comprise one or more components that are useful forpracticing at least one embodiment of the methods disclosed in thepresent teachings, or are produced through practice of at least oneembodiment of the methods disclosed in the present teachings. Thecompositions are not limited in their physical form, but are typicallysolids or liquids, or combinations of these. Furthermore, thecompositions may be present in any suitable environment, including, butnot limited to, reaction vessels (e.g., microfuge tubes, PCR tubes,plastic multi-well plates, microarrays), vials, ampoules, bottles, bags,and the like. In situations where a composition comprises a singlesubstance according to the present teachings, the composition willtypically comprise some other substance, such as water or an aqueoussolution, one or more salts, buffering agents, and/or biologicalmaterial. Compositions of the present teachings may comprise one or moreof the components of the present teachings, in any ratio or form.Likewise, they may comprise some or all of the reagents or moleculesnecessary for cross-linking of genomic DNA and protein, ligation ofhalf-adaptors and/or digested DNA, digestion of the cross-linked productand/or DNA template, amplification of the ligation products, orcombinations thereof. Thus, the compositions may comprise ATP, magnesiumor manganese salts, nucleotide triphosphates, and the like. They alsomay comprise some or all of the components necessary for generation of asignal from a labeled nucleic acid.

A composition of the present teachings may comprise one or morehalf-adaptors, for example, two or more half-adaptors. The half-adaptorsmay be any half-adaptor according to the present teachings, in anynumber of copies, any amount, or any concentration. The practitioner caneasily determine suitable amounts and concentrations based on theparticular use envisioned at the time. Thus, a composition according tothe present teachings may comprise a single half-adaptor. On the otherhand, it may comprise two or more half-adaptors, each of which may havethe same or a different sequence, or have the same or a different labelor capability for labeling, from all others in the composition.Non-limiting examples of compositions of the present teachings includecompositions comprising two or more half-adaptors and a samplecontaining or suspected of containing a cross-linked product includinggDNA and protein. Other non-limiting examples include compositionscomprising one or more half-adaptors, a sample containing or suspectedof containing a gDNA-protein cross-linked product, and at least oneligase, which is capable under the appropriate conditions, of catalyzingthe ligation of a half-adaptor to the cross-linked product and/or toanother half-adaptor. Yet other non-limiting examples of compositionsinclude those comprising two or more half-adaptors, a sample containingor suspected of containing a cross-linked product, at least one ligase,and at least one restriction endonuclease. Yet other non-limitingexamples of compositions include those comprising two or morehalf-adaptors, a sample containing or suspected of containing across-linked product, at least one ligase, at least one restrictionendonuclease, and at least one amplification primer. Yet othernon-limiting examples of compositions include those comprising two ormore half-adaptors, a sample containing or suspected of containing across-linked product, at least one ligase, at least one restrictionendonuclease, at least one amplification primer, and at least onepolymerase, which is capable under the appropriate conditions ofcatalyzing the polymerization of at least one amplification primer toform a polynucleotide. In certain embodiments, the compositions compriselabels or members of a labeling system.

Alternatively, a composition of the present teachings may comprise alysis solution comprising one or more detergents as disclosed herein.Some non-limiting examples of compositions include those comprising alysis solution comprising a combination of two or more detergents and asample containing or suspected of containing a cross-linked product. Yetother non-limiting examples of compositions include those comprising alysis solution comprising a combination of two or more detergents, asample containing or suspected of containing a cross-linked product anda restriction endonuclease. Yet other non-limiting examples ofcompositions include those comprising a lysis solution comprising acombination of two or more detergents, a sample containing or suspectedof containing a cross-linked product, a restriction endonuclease and twoor more half-adaptors. Yet other non-limiting examples of compositionsinclude those comprising a lysis solution comprising a combination oftwo or more detergents, a sample containing or suspected of containing across-linked product, a restriction endonuclease, two or morehalf-adaptors, and a ligase.

Alternatively, a composition of the present teachings may comprise oneor more ligation products of two half-adaptors. The ligation product maybe provided as the major substance in the composition, as when purifiedin a purified or partially purified form, or may be present as aminority of the substances in the composition. The ligation product maybe provided in any number of copies, in any amount, or at anyconcentration in the composition, advantageous amounts being easilyidentified by the practitioner for each particular purpose to which theligation product will be applied. In certain embodiments, thecomposition comprises agarose, polyacrylamide, or some other polymericmaterial that is suitable for isolating or purifying, at least to someextent, nucleic acids. In certain embodiments, the composition comprisesnylon, nitrocellulose, or some other solid support to which nucleicacids can bind. In some other embodiments, the composition comprisesmagnetic beads to which nucleic acids can bind. In some embodiments, thecompositions comprise at least one label or member or a labeling system.Two or more different ligation products may be present in a singlecomposition.

Alternatively, a composition of the present teachings may comprise oneor more amplification primers. The primer may be provided as the majorcomponent of the composition, such as in a purified or partiallypurified state, or may be a minor component. The primer may be anyamplification primer according to the present teachings, in any numberof copies, in any amount, or at any concentration. The practitioner caneasily determine suitable amounts and concentrations based on theparticular use envisioned at the time. In certain embodiments, acomposition of the present teachings may comprise one or more bridgeoligos. The bridge oligo may be provided as the major component of thecomposition, such as in a purified or partially purified state, or maybe a minor component. The bridge oligo may be any amplification primeraccording to the present teachings, in any number of copies, in anyamount, or at any concentration. The practitioner can easily determinesuitable amounts and concentrations based on the particular useenvisioned at the time.

Alternatively, a composition of the present teachings may comprise anamplification product. The amplification product may be any nucleic acidthat is derived (or has ultimately been produced) from a target DNAthrough practice of the methods of the present teachings, where themethod includes the step of amplification of the ligation product. Aswith other compositions comprising nucleic acids of the presentteachings, compositions comprising an amplification product may compriseit in any number of copies, amount or concentration. The amplificationproduct may be provided as the major substance in the composition, aswhen provided in a purified or partially purified form, or may bepresent as a minority of the substances in the composition. Non-limitingexamples of compositions of the present teachings include compositionscomprising an amplification product and a sample containing a targetDNA. Other non-limiting examples include compositions comprising anamplification product and at least two amplification primers. Othernon-limiting examples include those in which the composition comprisesan amplification product and at least one polymerase. Yet othernon-limiting examples include compositions comprising an amplificationproduct and at least one member of a labeling system. Yet othernon-limiting examples include compositions comprising an amplificationproduct and at least one ligase. Other non-limiting examples includecompositions comprising an amplification product and a ligation product.Further non-limiting examples include compositions comprising a targetDNA, a digested DNA and/or a cross-linked product, at least onehalf-adaptor, at least one ligase, at least one ligation product, atleast one amplification primer, at least one restriction endonuclease,at least one polymerse, at least one bridge oligo and an amplificationproduct. In certain embodiments, the composition comprises agarose,polyacrylamide, or some other polymeric material that is suitable forisolating or purifying, at least to some extent, nucleic acids. Incertain embodiments, the composition comprises nylon, nitrocellulose, orsome other solid support to which nucleic acids can bind. In some otherembodiments, the composition comprises magnetic beads to which nucleicacids can bind. In some embodiments, the compositions comprise at leastone label or member or a labeling system. Two or more different ligationproducts may be present in a single composition.

Compositions of the present teachings may comprise one or more nucleicacid polymerase. The polymerase may be any polymerase known to thoseskilled in the art as being useful for polymerizing a nucleic acidmolecule from a primer using a strand of nucleic acid as a template forincorporation of nucleotide bases. Thus, it may be, for example, Thermusthermophilus (Tth) DNA polymerase, Thermus aquaticus (Taq) DNApolymerase, Thermotoga neopolitana (Tne) DNA polymerase, Thermotogamaritima (Tma) DNA polymerase, Thermococcus litoralis (Tli or VENT™) DNApolymerase, Pyrococcus furiosus (Pfu) DNA polymerase, DEEPVENT™ DNApolymerase, Pyrococcus woosii (Pwo) DNA polymerase, Bacillussterothermophilus (Bst) DNA polymerase, Bacillus caldophilus (Bca) DNApolymerase, Sulfobus acidocaldarius (Sac) DNA polymerase, Thermoplasmaacidophilum (Tac) DNA polymerase, Thermus flavus (Tfl/Tub) DNApolymerase, Thermus ruber (Tru) DNA polymerase, Thermus brockianus(DYNAZYME™) DNA polymerase, Methanobacterium thermoautotrophicum (Mth)DNA polymerase, mycobacterium DNA polymerase (Mtb, Mlep), and mutants,and variants and derivatives thereof. Generally, any type I DNApolymerase may be used in accordance with the present teachings althoughother DNA polymerases may be used including, but not limited to, typeIII or family A, B, C etc., DNA polymerases. In addition, the nucleicacid polymerases may be mesophilic or thermophilic. Exemplary mesophilicDNA polymerases include T7 DNA polymerase, T5 DNA polymerase, Klenowfragment DNA polymerase, DNA polymerase III and the like. Exemplarythermostable DNA polymerases include Taq, Tne, Tma, Pfu, Tfl, Tth,Stoffel fragment, VENT™ and DEEPVENT™ DNA polymerases, and mutants,variants and derivatives thereof.

Compositions of the present teachings may comprise one or more ligase.The ligase may be any ligase known to those skilled in the art as beinguseful for ligating a nucleic acid molecule. Thus, it may be, forexample, T4 DNA ligase, Tfi DNA ligase, DNA ligase I, DNA ligase II, DNAligase III, DNA ligase IV, and small footprint DNA ligases.

According to another embodiment of the present teachings, the chromosomeconformation analysis methods disclosed herein may be used in diagnosticand/or prognostic methods for identifying diseases or in determiningpatient response to treatment with certain drugs, medications or methodsof therapy. An exemplary condition that can be associated with the threedimensional structure of chromatin is cancer. Thus, the presentteachings provide a method of diagnosing susceptibility to a cancer orprognosis of outcome for treatment of cancer.

The prognostic methods of the present teachings are useful fordetermining if a patient is at risk for recurrence. Cancer recurrence isa concern relating to a variety of types of cancer. For example, ofpatients undergoing complete surgical removal of colon cancer, 25-40% ofpatients with stage II colon carcinoma and about 50% of patients withstage III colon carcinoma experience cancer recurrence. One explanationfor cancer recurrence is that patients with relatively early stagedisease, for example, stage II or stage III, already have small amountsof cancer spread outside of the affected organ that were not removed bysurgery. These cancer cells, referred to as micrometastases, cannottypically be detected with currently available tests.

The prognostic methods disclosed herein can be used to identifysurgically treated patients likely to experience cancer recurrence sothat they can be offered additional therapeutic options, includingpreoperative or postoperative adjuncts such as chemotherapy, radiation,biological modifiers and other suitable therapies. The methods areespecially effective for determining the risk of metastasis in patientswho demonstrate no measurable metastasis at the time of examination orsurgery.

The prognostic methods according to certain embodiments also are usefulfor determining a proper course of treatment for a patient havingcancer. A course of treatment refers to the therapeutic measures takenfor a patient after diagnosis or after treatment for cancer. Forexample, a determination of the likelihood for cancer recurrence,spread, or patient survival, can assist in determining whether a moreconservative or more radical approach to therapy should be taken, orwhether treatment modalities should be combined. For example, whencancer recurrence is likely, it can be advantageous to precede or followsurgical treatment with chemotherapy, radiation, immunotherapy,biological modifier therapy, gene therapy, vaccines, and the like, oradjust the span of time during which the patient is treated. Asdescribed herein, the diagnosis or prognosis of cancer state can beassociated with the three-dimensional conformation of the patient'schromatin structure. Recent studies suggest that epigenetic alterationsmay be the key initiating events in some forms of cancers and globalchanges in the epigenome are a hallmark of cancers.

Exemplary cancers that may be evaluated using a method as disclosedherein include, but are not limited to hematoporetic neoplasms, AdultT-cell leukemia/lymphoma, Lymphoid Neoplasms, Anaplastic large celllymphoma, Myeloid Neoplasms, Histiocytoses, Hodgkin Diseases (HD),Precursor B lymphoblastic leukemia/lymphoma (ALL), Acute myclogenousleukemia (AML), Precursor T lymphoblastic leukemia/lymphoma (ALL),Myclodysplastic syndromes, Chronic Mycloproliferative disorders, Chroniclymphocytic leukemia/small lymphocytic lymphoma (SLL), ChronicMyclogenous Leukemia (CML), Lymphoplasmacytic lymphoma, PolycythemiaVera, Mantle cell lymphoma, Essential Thrombocytosis, Follicularlymphoma, Myelofibrosis with Myeloid Metaplasia, Marginal zone lymphoma,Hairy cell leukemia, Hemangioma, Plasmacytoma/plasma cell myeloma,Lymphangioma, Glomangioma, Diffuse large B-cell lymphoma, KaposiSarcoma, Hemanioendothelioma, Burkitt lymphoma, Angiosarcoma, T-cellchronic lymphocytic leukemia, Hemangiopericytoma, Large granularlymphocytic leukemia, head & neck cancers, Basal Cell Carcinoma, Mycosisfungoids and sezary syndrome, Squamous Cell Carcinoma, Ceruminoma,Peripheral T-cell lymphoma, Osteoma, Nonchromaffin Paraganglioma,Angioimmunoblastic T-cell lymphoma, Acoustic Neurinoma, Adenoid CysticCarcinoma, Angiocentric lymphoma, Mucoepidermoid Carcinoma, NK/T-celllymphoma, Malignant Mixed Tumors, Intestinal T-cell lymphoma,Adenocarcinoma, Malignant Mesothelioma, Fibrosarcoma, Sarcomotoid Typelung cancer, Osteosarcoma, Epithelial Type lung cancer, Chondrosarcoma,Melanoma, cancer of the gastrointestinal tract, olfactory Neuroblastoma,Squamous Cell Carcinoma, Isolated Plasmocytoma, Adenocarcinoma, InvertedPapillomas, Carcinoid, Undifferentiated Carcinoma, Malignant Melanoma,Mucoepidermoid Carcinoma, Adenocarcinoma, Acinic Cell Carcinoma, GastricCarcinoma, Malignant Mixed Tumor, Gastric Lymphoma, Gastric Stromal CellTumors, Amenoblastoma, Lymphoma, Odontoma, Intestinal Stromal Celltumors, thymus cancers, Malignant Thymoma, Carcinids, Type I (Invasivethymoma), Malignant Mesethelioma, Type II (Thymic carcinoma), Non-mucinproducing adenocarcinoma, Squamous cell carcinoma, Lymph epithelioma,cancers of the liver and biliary tract, Squamous Cell Carcinoma,Hepatocellular Carcinoma, Adenocarcinoma, Cholangiocarcinoma,Hepatoblastoma, papillary cancer, Angiosarcoma, solid Bronchioalveolarcancer, Fibrolameller Carcinoma, Small Cell Carcinoma, Carcinoma of theGallbladder, Intermediate Cell carcinoma, Large Cell Carcinoma, SquamousCell Carcinoma, Undifferentiated cancer, cancer of the pancreas, cancerof the female genital tract, Squamous Cell Carcinoma,Cystadenocarcinoma, Basal Cell Carcinoma, Insulinoma, Melanoma,Gastrinoma, Fibrosarcoma, Glucagonamoa, Intaepithelial Carcinoma,Adenocarcinoma Embryonal, cancer of the kidney, Rhabdomysarcoma, RenalCell Carcinoma, Large Cell Carcinoma, Nephroblastoma (Wilm's tumor),Neuroendocrine or Oat Cell carcinoma, cancer of the lower urinary tract,Adenosquamous Carcinoma, Urothelial Tumors, Undifferentiated Carcinoma,Squamous Cell Carcinoma, Carcinoma of the female genital tract, MixedCarcinoma, Adenoacanthoma, Sarcoma, Small Cell Carcinoma,Carcinosarcoma, Leiomyosarcoma, Endometrial Stromal Sarcoma, cancer ofthe male genital tract, Serous Cystadenocarcinoma, MucinousCystadenocarcinoma, Sarcinoma, Endometrioid Tumors, SperetocyticSarcinoma, Embyonal Carcinoma, Celioblastoma, Choriocarcinoma, Teratoma,Clear Cell Carcinoma, Leydig Cell Tumor, Unclassified Carcinoma, SertoliCell Tumor, Granulosa-Theca Cell Tumor, Sertoli-Leydig Cell Tumor,Disgerminoma, Undifferentiated Prostatic Carcinoma, Teratoma, DuctalTransitional carcinoma, breast cancer, Phyllodes Tumor, cancer of thebones joints and soft tissue, Paget's Disease, Multiple Myeloma, InsituCarcinoma, Malignant Lymphoma, Invasive Carcinoma, Chondrosacrcoma,Mesenchymal Chondrosarcoma, cancer of the endocrine system,Osteosarcoma, Adenoma, Ewing Tumor, endocrine Carcinoma, Malignant GiantCell Tumor, Meningnoma, Adamantinoma, Cramiopharlingioma, MalignantFibrous Histiocytoma, Papillary Carcinoma, Histiocytoma, FollicularCarcinoma, Desmoplastic Fibroma, Medullary Carcinoma, Fibrosarcoma,Anoplastic Carcinoma, Chordoma, Adenoma, Hemangioendothelioma,Memangispericytoma, Pheochromocytoma, Liposarcoma, Neuroblastoma,Paraganglioma, Histiocytoma, Pineal cancer, Rhabdomysarcoms,Pineoblastoma, Leiomyo sarcoma, Pineocytoma, Angiosarcoma, skin cancer,cancer of the nervous system, Melanoma, Schwannoma, Squamous cellcarcinoma, Neurofibroma, Basal cell carcinoma, Malignant Periferal NerveSheath Tumor, Merkel cell carcinoma, Sheath Tumor, Extramamary Paget'sDisease, Astrocytoma, Paget's Disease of the nipple, FibrillaryAstrocytoma, Glioblastoma Multiforme, Brain Stem Glioma, CutaneousT-cell lymphoma, Pilocytic Astrocytoma, Xanthorstrocytoma,Histiocytosis, Oligodendroglioma, Ependymoma, Gangliocytoma, CerebralNeuroblastoma, Central Neurocytoma, Dysembryoplastic NeuroepithelialTumor, Medulloblastoma, Malignant Meningioma, Primary Brain Lymphoma,Primary Brain Germ Cell Tumor, cancers of the eye, Squamous CellCarcinoma, Mucoepidermoid Carcinoma, Melanoma, Retinoblastoma, Glioma,Meningioma, cancer of the heart, Myxoma, Fibroma, Lipoma, PapillaryFibroelastoma, Rhasdoyoma, or Angiosarcoma among others.

One embodiment provides methods for diagnosing the occurrence of cancerin a patient or a patient at risk for cancer. The method involves (a)determining the epigenetic interactions of the genomic DNA using any oneof the chromosomal conformation analysis methods disclosed herein, and(b) comparing the epigenetic interactions to a reference sample (i.e., asample isolated from a healthy patient, tissue or cell), wherein one ormore different epigenetic interactions correlates with presence ofcancer in the patient.

Another embodiment provides methods for determining a prognosis forsurvival for a cancer patient. One method involves (a) determining theepigenetic interactions of the genomic DNA using any one of thechromosomal conformation analysis methods disclosed herein from a sampleobtained from the cancer patient, and (b) comparing the epigeneticinteractions to a reference sample (i.e., a sample isolated from ahealthy patient, tissue or cell), wherein one or more differentepigenetic interactions correlates with increased survival of thepatient.

Yet another embodiment provides a method for monitoring theeffectiveness of a course of treatment for a patient with cancer. Themethod involves (a) determining the epigenetic interactions of thegenomic DNA using any one of the chromosomal conformation analysismethods disclosed herein from a sample from the cancer patient prior totreatment, and (b) comparing the epigenetic interactions to a samplefrom the patient after treatment, whereby comparison of the epigeneticinteractions after treatment indicates the effectiveness of thetreatment.

Yet another embodiment provides a method that may be used to determinethe prognosis of disease free survival or overall survival. As usedherein, the term “disease-free survival” refers to the lack ofrecurrence of symptoms such as, in the case of cancer, lack of tumorrecurrence and/or spread and the fate of a patient after diagnosis, forexample, a patient who is alive without tumor recurrence. The phrase“overall survival” refers to the fate of the patient after diagnosis,regardless of whether the patient has a recurrence of symptoms such as,in the case of cancer, tumor recurrence. Tumor recurrence refers tofurther growth of neoplastic or cancerous cells after diagnosis ofcancer. Particularly, recurrence can occur when further cancerous cellgrowth occurs in the cancerous tissue. Tumor spread refers todissemination of cancer cells into local or distant tissues and organs,for example, during tumor metastasis. Tumor recurrence, in particular,metastasis, is a significant cause of mortality among patients who haveundergone surgical treatment for cancer. Therefore, tumor recurrence orspread is correlated with disease-free and overall patient survival.

Similar methods to those exemplified above for cancer may be used todiagnose or prognose other conditions. For example, the methods can beuseful for diagnosing early-onset inherited Parkinson's disease or otherdiseases that arise due to aberrant gene expression. Thus, the stepsexemplified above for cancer can also be used to diagnose these otherdiseases, to prognose survival rate or to monitor effectiveness of acourse of treatment.

While the present teachings have been described in terms of theseexemplary embodiments, the skilled artisan will readily understand thatnumerous variations and modifications of these exemplary embodiments arepossible without undue experimentation. All such variations andmodifications are within the scope of the current teachings. Aspects ofthe present teachings may be further understood in light of thefollowing examples, which should not be construed as limiting the scopeof the teachings in any way.

EXAMPLES Example 1 Restriction and Ligation Control Assays

Control assays were developed to provide quantitative measurement ofimportant steps in the methods disclosed herein. qPCR control assays forthe monitoring of efficiencies of restriction digestion and ligationsteps were designed and tested (see, FIG. 2). Since digestion byrestriction endonucleases generates new ends and ligation removes them,restriction and ligation efficiency may be monitored with one pair ofassays. The generation of a restricted end (generated by restrictionendonuclease digestion) reveals the efficiency of restriction digestionwhile the “disappearance” of a restricted end reflects ligationefficiency.

The forward primer of Assay1 was selected from a sequence upstream ofthe restriction site while the reverse primer was selected from asequence downstream of the restriction site. It amplifies and detectsthe intact (undigested) template. It has inherently higher sensitivityand precision for high digestion efficiency (>50%) but lower sensitivityfor low digestion efficiency (<50%).

However, restriction digestion is greatly hindered by the cross-linkingof DNA with proteins, which reduces digestion efficiency drastically. Asa result, assays that are more sensitive and precise for low digestionefficiency are needed. The embodiments of the methods disclosed hereininclude a new assay to detect the newly generated digested ends by usinga 3′-blocked oligonucleotide (herein denoted a “bridge oligo”) to annealto the sequence at the restriction site. The bridge oligo may be copiedthorough the extension of the newly generated 3′ end at the restrictionrecognition site.

The bridge oligo contains a 3′ sequence to anneal to the digested endand a 5′-region to hybridize with TaqMan® probe and reverse primer. Inaddition to the bridge oligo, the “bridged assay” consists of a forwardprimer picked from the genomic locus upstream of the bridge annealingregion and the probe and reverse primer picked from the 5′-region of thebridge oligo. The bridged PCR detects only the digested end which cananneal and copy the bridge oligo. In addition, the bridged assaydetected digested DNA sequences down to 1%, making it uniquely suitablefor the measurement of low digestion efficiency typical of 3C-basedmethods. Paired with standard qPCR assays that use primers spanning therestriction site, the bridged assay provided accurate and precisequantitation of restriction and ligation efficiencies. Bydesign-of-experiments, the restriction and ligation efficiencies weredemonstrated as highly predictive of 3C library quality. Control assayswere successfully designed and validated for common restriction enzymes(e.g. EcoR I, Bgl II, Tsp509 I, Hind III, Sau3A I, Dpn II and Alu I).

The restriction and ligation control assays were picked from a genedesert region, which is expected to have few changes among differentcells and cell stages (see, FIG. 8). The target sequences for both EcoRI and Hind III were centered at a region that consists of the tworestriction sites from human ENCODE gene desert region, ENr313 inchromosome 16 (>gi|42655553|ref|NT_(—)086350.2|ENr313 Homo sapienschromosome 16 sequence, ENCODE region ENr313). The combination of theassays was used to monitor both restriction and ligation.

Example 2 Calculation of Digestion and Ligation Efficiency (FIG. 9)

Digestion and ligation efficiency may be calculated by qPCR Ct values ofthe control assays (i.e., Assays 1 and 2, see FIG. 2). The two qPCRassays were combined to provide high sensitivity and precision inefficiency measurement.

Assay 1 was designed to quantitate undigested (residual) template. ItsCt follows a standard qPCR response:Ct ₁ =−k ₁*log(C ₀*% Undigested)+Ct ₁₀  (Formula 1)

wherein Ct₁ is the Ct value of Assay 1 in a test, k₁ is the absolutevalue of the standard curve slope, C₀ is total template concentration, %Undigested is the percentage of undigested template, and C₁₀ is theintercept of standard curve.

Assay 2, called a bridge assay, detects the amount of digested template.Its Ct response to digestion efficiency follows a standard qPCRequation:Ct ₂ =−k ₂*log(C ₀*% Digested)+Ct ₂₀  (Formula 2)

wherein Ct₂ is Ct value of Assay 2 in a test, k₂ is the absolute valueof the standard curve slope, C₀ is total template concentration, %Digested is the percentage of undigested template at the assay site, andC₂₀ is the intercept of standard curve. Since % Undigested+%Digested=100%, % Undigested=1−% Digested.

The difference of Ct between Assay 2 and Assay1 may be calculated asfollows:ΔCt=Ct ₂ −Ct ₁ =−k ₂*log(C ₀*% Digested)+Ct ₂₀ ]−[−k ₁*log(C ₀*%Undigested)+Ct ₁₀]=log [(% Undigested/% Digested)^(k ₁ /k ₂)]+(Ct ₂₀ −Ct₁₀)=k ₁*log(% Undigested/% Digested)+ΔCt ₀−(k ₁ −k ₂)*log(% Undigested/%Digested) when k ₁ =k ₂ =k, ΔCt=k*log(% Undigested/% Digested)+ΔCt₀.  (Formula 3)

As a result, % Digested=1/(1+10^[(ΔCt−ΔCt₀)/k]. The values of k and Ct₀can be measured by plotting ΔCt versus log(% Undigested/% Digested).

Titration of the % Digested using EcoR I or Hind III digested genomicDNA validated the linearity of ΔCt(Assay2−Assay1) vs. Log(% Undigested/%Digested). A template amount of 0.31 ng/ul to 10 ng/μl in PCR did notshift the curve.

Final equations for restriction efficiency calculation:EcoR I: % Digestion=1/[1+10^(ΔCt−4.404)/3.28]  (Formula 5)Hind III: % Digestion=1/[1+10^(ΔCt−5.66)/3.015]  (Formula 6)

A standard ΔΔCt method may be used for ligation efficiency calculation.% Ligation=1−½^(ΔΔCt), where ΔΔCt=ΔCt(Ct ₂ −Ct _(r))_(Digested) −ΔCt(Ct₂−Ct_(r))_(Ligated). Ct_(r): Ct of reference assay.  (Formula 7)

RNase P and other copy number reference assays may be used as long asthey do not contain EcoR I and Hind III recognition sites and are ofsingle copy per genome.

Example 3 Optimization of Ligation Volume and Reducing Between-CellLigation (FIG. 10)

Cells were cross-linked with 1% formaldehyde, lysed and digested withAlu I (400 U/5 million cells) in NEBuffer 2 (New England Biolabs,Ipswich, Mass.) for 1 hour at 37° C. Alu I was then heat inactivated at65° C. for 20 min. Digested cells were spun down and washed withNEBuffer2. For filling-up and A-tailing, 5×10⁶ cells were mixed with 15μl 10×NEBuffer 2, 1.5 μl 100 mM dNTP, 1 μl Klenow (exo⁻, 50 U/μl) in afinal volume of 150 μl and incubated at 37° C. for 1 hr. Cells were thenspun down and washed with NEBuffer 3. Cells were resuspended in 1× T4ligase buffer, pooled and split into two reactions. Each reaction wasligated to a half-adaptor at the following conditions and incubated at16° C. for 1 hr:

Half-adaptor (50 μM)  4 μl 5× Invitrogen Ligase Buffer  60 μl T4 Ligase(1 mg/ml)  15 μl H₂O 221 μl Total Volume: 300 μl

The cells were spun down and washed twice with 500 μl 1× T4 ligasebuffer. The separately ligated cells were resuspended in 1× T4 ligasebuffer and mixed in equal portions, ligated at concentrations of 2.5,25, 75, and 122.5 ng/μl gDNA equivalent (assuming a diploid genome) withdifferent amounts of T4 ligase and varying the ligation time to evaluatethe effects of cell concentration.

Ligation between cells was quantitated using TaqMan® assays designed tospecifically amplify and detect sequences with the full adaptor(ligation product of two half-adaptors) located between two Alurepetitive sequences. Ligation event numbers were calculated by Ctvalues after normalization with RNase P (see, FIG. 10).

Ligation capacity (ligase concentration×ligation time) was the dominantfactor for both long range interaction ligation and between-cellligation, accounting for up to 70% of the variations in between-cellligations. Reduction of the ligation volume by 49-fold only doubled thebetween-cell ligation events per cell regardless of ligation capacity.The increase of between-cell ligation was paralleled with increasedligation efficiency and was increased in a dose-dependent manner by cellconcentration (a 4-fold increase by a 30-fold cell increase) regardlessof the ligation capacity. Optimization of ligation amount and ligationtime was more efficient in reducing non-specific ligation.

Example 4 Assays for Long-Range Interaction Detection (FIGS. 11-14)

To monitor interaction capture efficiencies, TaqMan® assays weredesigned to detect cross-ligated DNA sequences. 3C targets between humanβ-globin locus control region (LCR) and human β-globin (HBG), which areseparated by about 40 kb, were used to monitor the interaction detectionfor euchromatin region and assays for heterochromatin were selectedbetween two nearby sequences in a gene desert (see FIG. 8). Optimallysis buffer and conditions were selected after comparison of differentdetergents and their combinations. Reagents and conditions fordigestion, ligation and purification were optimized through systematicdesign-of-experiments and titrations. Cells of K562 and GM06990 wereused at 1 to 10 million per reaction for the optimization. Theexperimental design is outlined below.

Long-Range Interaction Between LCR and β-Globin Experimentals:

Sample:

-   -   Bead purified 3C library of K562 cell lines    -   Pooled DNA from ligation    -   Pre-amplified vs. non-amplified comparison    -   CNV RNase P assays used for template quantitation with Jurkat        gDNA (NEB) as standard

LCR Assays:

-   -   Loci 1 to 36 (out of 107)    -   3 LCR cutting sites: HS1-2 (#26), HS4-5 (#27) and 5′-HS5 (#28)    -   Combinations: #26Fw+#1-36 Fw; #27Fw+#1-36Fw; #27Fw+#1-36Rv;        #28Fw+#1-36Fw; #27Rv+#1-36Rv; #27Rv+#1-36Fw

Gene Desert Assays:

-   -   #24Fw+#14-77Fw; #74Fw+#14-77Fw 3 μl/10 μl PCR

Data Analysis:

-   -   Ct correction with BAC control library    -   PCR efficiency vs. Ct-based correction    -   LCR looping model

qPCR assays were designed for 107 EcoR I sites around LCR and β-globinfollowing TaqMan® assay criteria for human LCR sequence(>gi|42655566|ref|NT_(—)086365.3|ENm009 Homo sapiens chromosome 11sequence, ENCODE region ENm009). These assays were used to quantitateinteraction frequencies between LCR with the β-globin genes (see, FIGS.11 and 12). BAC clones were used to normalize qPCR efficiency of theassays. BAC DNA covering the targeted LCR region were isolated and mixedin equal ratio followed by restriction digestion by EcoR I or Hind IIIand ligation by T4 ligase.

Two assays that detected the strongest long-range interactions wereselected: one between LCR HS 1-2 (F26) and HBG1 (F14) and anotherbetween LCR HS 4-5 (F27) and HBG1 (F14). These assays were validated forboth EcoR I and Hind III 3C libraries. F26 interaction with F14 was fourfold higher than F27 (FIG. 13 and data not shown), indicating thedifference in their distance to F14. Variations in the response of thesetwo assays to the process conditions disclosed herein are expected toreveal distance-dependence of capture efficiency changes. Athree-dimensional schematic diagram for the genomic interactions betweenLCR and HBG based on the assays described above is shown in FIG. 14.

Example 5 Capturing LCRE Interaction (FIG. 15)

TaqMan® assays were designed with a forward primer and probe targetingthe Eco RI upstream sequence located between LCR HS4 and LCR HS5 and areverse primer from the HBG1 region. PCR reactions were run on a 7900HTSreal-time PCR machine (Life Technologies, Foster City, Calif.) usingTaqMan® Gene Expression Master Mix (Life Technologies, Foster City,Calif.) at standard cycling conditions to generate the amplificationplot (FIG. 16). PCR products were loaded on a 4% agarose gel and run onan E-gel system (Life Technologies, Carlsbad, Calif.). Sequencesamplified from templates without adaptors were detected at ˜120 bp andthose from templates with full adaptors were detected at ˜150 bp (FIG.16).

Cells of K562 were processed using the chromosome interaction methodsdisclosed herein until the DNA purification step. Linear half-adaptorswere used for gel lanes 1-6 at the indicated concentration. Loopedadaptors were used for gel lanes 7 and 8. Lane 9 shows the PCR productfor a 3C library (see, FIG. 15).

Example 6 Complete 3C Library Generation (FIG. 16)

DNA libraries were generated according to the methods disclosed hereinuntil the library amplification step. PCR products were analyzed on 4%agarose gels. Expected products were detected at ˜156 bp. Lanes 1-8 ofFIG. 16 correspond to lane 9 in FIG. 15.

Example 7 Identification of Epigenetic Networks in Breast Cancer (FIGS.17-21)

Epigenetic mechanisms are essential for normal development andmaintenance of normal gene expression pattern in many organismsincluding humans. Recent studies suggest that epigenetic alterations maybe the key initiating events in some forms of cancers and global changesin the epigenome are a hallmark of cancers. Phospolipase D (PLD)catalyzes the hydrolysis of phosphatidylcholine to generate the lipidsecond messenger, phosphatidic acid (PA), and choline and regulatesmultiple cellular pathways. Elevated PLD1 has been demonstrated topromote cell proliferation and has been associated with the progressionand metastasis of multiple cancers. In certain embodiments, thechromosomal conformation methods described herein may be used toelucidate the changes in chromatin organization, DNA methylation andmiRNA expression that underlies the aberrant expression level of PLD1 inbreast cancers. Differences in PLD1 expression and the epigeneticnetwork between tamoxifen responsive (MCF-7) and non-responsive(MDA-MB-231) breast cancer cell lines were compared to associateepigenetic changes with their cancer phenotypes. Effects of DNAdemethylation and histone acetylation induced by the methyltransferaseinhibitor 5-aza-2′-deoxycytidine and the histone deacetylase inhibitorTrichostatin A were also investigated to provide insights into themolecular mechanisms of epigenetic drugs.

Epigenetic mechanisms that modify chromatin structure can be dividedinto four main categories: 1) DNA methylation, 2) covalent histonemodifications and noncovalent mechanisms such as incorporation ofhistone variants, 3) nucleosome remodeling and 4) non-coding RNAs whichinclude miRNAs. The role of DNA methylation and histone modifications incancer initiation and progression is well established; however, thechanges in chromatin structure that accompany DNA methylation andhistone modifications are less well understood.

Two breast cancer cell lines, MCF-7 and MDA-MB-231 were obtained fromthe American Type Culture Collection (ATCC). Drugs used to treat cellssuch as 5-Aza-2′-deoxycytidine, Trichostatin A, Phorbol 12-myristate13-acetate (tumor promoter, PKC activator), 4-hydroxytamoxifen(Tamoxifen) and β-estradiol were purchased from Sigma (St. Louis, Mo.).Assays used for miRNA profiling, methylation, and Taqman® geneexpression were obtained from Life Technologies (Carlsbad, Calif.). Thelist of breast cancer markers, assays and gene symbols used in thisexample are disclosed in Tables 2-4, below.

TABLE 2 List of Breast Cancer Markers Tumor Suppressors Cancer Type APCLung, colon, breast, gastric, liver BLC2 Bladder, colon, breast,prostate HS3ST2 Pancreas, lung, breast, colon, skin, gall bladder IGF2AS Colon PTEN Skin, lung, breast RPRM Esophageal, lung, pancreas SCGB3A1Prostate, breast, lung, nasopharyngeal, pancreas SYK Breast, gastric,liver ZMYND10 Cervical, lung, nasopharyngeal, brain, liver PLD1Overexpressed in many cancers

TABLE 3 Assays and Their Targets Assay Name Target Functional PathwayFunction hsa-miR-206 ESR-1 ER signaling Tumor suppressor miRNAmo-miR-17-5p E2F1, AlB1, CCND1 Proliferation Tumor suppressor miRNAhsa-miR-125a-3p Her2, Her3 Anchorage dependent Tumor suppressor growthmiRNA hsa-miR-125a-5p Her2, Her3 Anchorage dependent Tumor suppressorgrowth miRNA hsa-miR-200b BMI1, ZEB1, ZEB2 TGF-B signaling Tumorsuppressor miRNA hsa-let-7a H-ras, HMGAS2, Proliferation, Tumorsuppressor LIN28, PEBP1 differentiation miRNA hsa-miR-34a CCND1, DCK6,DNA damage, Tumor suppressor E2F3, MYC proliferation miRNA hsa-miR-31FZD3, ITGA5, M- metastasis Tumor suppressor RIP, MMP16, RDX, miRNA RHOAhsa-miR-21 BCL-2, TPM1, Apoptosis Oncogenic miRNAs PDCD4, PTEN, MASPINhsa-miR-155 RHOA TGF-B signaling Oncogenic miRNAs hsa-miR-10b HOXD10Metastasis Oncogenic miRNAs hsa-miR-373 CD44 Metastasis Oncogenic miRNAshsa-miR-16 Control Control Control RNU44 Control, Homo Control Controlsapiens RNU48 Control, Homo Control Control sapiens

TABLE 4 Gene Symbols Gene Symbols PLD1 (exon 15/16) MAPK3 PLD1 (exon1/2) CDKN1A ERAF PRKAR1A ESR1 PPM1D ESR2 PIK3CA TP53 APC CTNNBL1 BCL2TCF7L2 HS3ST2 KRAS IFG2AS BRMS1L PTEN PBQV1 RPRM AR SCGB3A1 BCAR4 SYKCAMK2B ZYMKD10 BRCA2 ACTB BRMS1L GAPDH

FIG. 17 shows a diagrammatic representation of PLD1 genes and the assaydesigns using the chromosomal conformation methods according to theembodiments disclosed herein. PLD1 Intron 1 and PLD1 Promoter 2 wereused as anchors to capture other downstream cis interactions across thewhole gene using the chromosome conformation methods disclosed herein.In FIGS. 18A and B, the x-axis shows the distance (kb) of the downstream(upstream) primers (EcoRI sites) to Intron 1 or Promoter 2. The y-axisshows the relative interaction frequencies based on qPCR Ct (ΔCt). Thelower the ΔCt, the higher the interaction frequency. Comparing theMDA-MB-231 results with those of MCF-7, the difference in the longdistance interaction frequency between these two cell lines is apparent.This difference could possibly explain the differences in drug responseof these two types of breast cancer patients.

The methylation (FIG. 19A) and expression (FIG. 19B) of PLD1 werecompared between the two cell lines. As can be seen in the figures, thePLD1 expression level for both Promoter 1 and Promoter 2 were compared.The expression ratio of MDA-MB-231 to MCF-7 for Promoter 1 alone is 10,and for Promoter 1 and 2 together is 5. Both cell lines are unmethylatedin this region (data not shown).

FIG. 20 shows a comparison of cancer marker genes and microRNAs for thetwo cell lines. FIG. 20A shows 12 cancer marker microRNAs including fouroncogenic and eight tumor suppressors which were compared between MCF-7and MDA-MB-231. Expression levels were normalized by RNU44 and RNU48.FIG. 20B shows the expression level of 30 cancer marker genes including14 tumor suppressors (10 of which were also compared for methylation)and 16 oncogenic genes were compared.

FIG. 21 shows a comparison of the methylation of CpGs (13 total) in theBCL-2 promoter region (350 bp from the transcription start) which showeddifferential methylation: 0% methylation in MCF-7 and 100% methylationin MDA-MB-231. BCL2 gene expression levels were compared resulting in analmost 8-fold higher expression in MCF-7, which is consistent with thehypermethylation in MDA-MB-231. In addition, oncogenic MiR21 wasexpressed about 5-fold higher in MCF-7.

Epigenetic alterations could explain the aberrant expression of manygenes which contribute to the alteration of metastasis. Comparison ofthe epigenetic changes in chromatin 3D structure of PLD1 genes, changesof methylation in breast cancer-related tumor suppressor genes andexpression level changes of microRNAs between two breast cancer cellslines was performed. In addition, the expression of metastasis and tumorsuppressor genes was also compared. The 3D structure of PLD1 indicatesthere are more interactions of both Intron 1 and Promoter 2 with othercis elements across the 215 kb gene in the less metastasized MCF-7 cellline. However, the expression level of PLD1 is much higher in moremetastasized MDA-MB 231 cells. 3D structure changes could potentiallyexplain the expression level changes which in turn, explain themetastasis changes. PLD1 3D structure changes upon epigenetic drugtreatments were also captured (data not shown). Another importantepigenetic modification, methylation, was also monitored. Between thetwo cell lines, most tumor suppressor genes showed hypermethylation inMDA-MB 231, which is consistent with most observations. Furthermore,gene expression level of promoters in the methylation study was alsomeasured. The data disclosed herein showed no absolute correlationbetween methylation and gene expression level changes. MicroRNAprofiling showed over expression of both oncogenic and tumor suppressormicroRNAs in MCF-7 in majority of microRNAs included in the study. Thecomparison of oncogenic MiR21 and its target gene BCL2 showed a negativecorrelation.

We claim:
 1. A method of determining the three-dimensional arrangementof the chromatin within a cell, comprising: a) isolating cells from abiological sample; b) incubating the cells with a cross-linking agent,thereby cross-linking proteins with DNA and forming a cross-linkedproduct; c) lysing the cells; d) digesting the DNA with a restrictionendonuclease; e) incubating the digested DNA with a phosphatase; f)ligating a first looped half-adaptor to the digested DNA thereby forminga first ligation product and a second looped half-adaptor to thedigested DNA thereby forming a second ligation product, wherein thefirst and second looped half-adaptors are different from each other andeach looped half-adaptor comprises a stem-loop structure complementaryto the stem-loop structure of the other half-adaptor, wherein the 5′ endof the stem of each looped half-adaptor comprises a 5′-overhang thatterminates in a 5′-phosphate group and is ligated to the digested DNA;g) heating the first and second ligation products thereby allowing thehalf-adaptors to anneal to each other; h) treating the product of stepg) with a kinase and a ligase to ligate the looped half-adaptors to thedigested DNA thereby forming a third ligation product; and i) analyzingthe third ligation product.
 2. The method according to claim 1, whereinthe analyzing step is performed using the polymerase chain reaction(PCR).
 3. The method according to claim 1, further comprising the stepof incubating the cross-linked product with a cross-linking quencher. 4.The method according to claim 1, wherein the lysing step comprisesincubating the cells with one or more detergents.
 5. The methodaccording to claim 4, wherein the one or more detergents are selectedfrom the group consisting of anionic detergents and non-ionicdetergents.
 6. The method according to claim 1, wherein thecross-linking agent comprises formaldehyde.